Substituted pyridines as selective cyclooxygenase-2 inhibitors

ABSTRACT

The invention encompasses the novel compound of Formula I as well as a method of treating COX-2 mediated diseases comprising administration to a patient in need of such treatment of a non-toxic therapeutically effective amount of a compound of Formula I. ##STR1## The invention also encompasses certain pharmaceutical compositions for treatment of COX-2 mediated diseases comprising compounds of Formula I.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 08/893,395filed on Jul. 11, 1997, now U.S. Pat. No. 5,861,419 granted on Jan. 19,1999, and U.S. application Ser. No. 09/181,887 filed on Oct. 29, 1998,now U.S. Pat. No. 6,001,843, and copending herewith, based uponProvisional Application Nos. 60/022,128 filed on Jul. 18, 1996,60/027,139 filed on Oct. 1, 1996 and 60/041,814 filed on Apr. 8, 1997,priority of which is claimed hereunder.

BACKGROUND OF THE INVENTION

This invention relates to methods of treating cyclooxygenase mediateddiseases and certain pharmaceutical compositions therefor.

Non-steroidal, antiinflammatory drugs exert most of theirantiinflammatory, analgesic and antipyretic activity and inhibithormone-induced uterine contractions and certain types of cancer growththrough inhibition of prostaglandin G/H synthase, also known ascyclooxygenase. Initially, only one form of cyclooxygenase was known,this corresponding to cyclooxygenase-1 (COX-1) or the constitutiveenzyme, as originally identified in bovine seminal vesicles. Morerecently the gene for a second inducible form of cyclooxygenase,cyclooxygenase-2 (COX-2) has been cloned, sequenced and characterizedinitially from chicken, murine and human sources. This enzyme isdistinct from the COX-1 which has been cloned, sequenced andcharacterized from various sources including the sheep, the mouse andman. The second form of cyclooxygenase, COX-2, is rapidly and readilyinducible by a number of agents including mitogens, endotoxin, hormones,cytokines and growth factors. As prostaglandins have both physiologicaland pathological roles, we have concluded that the constitutive enzyme,COX-1, is responsible, in large part, for endogenous basal release ofprostaglandins and hence is important in their physiological functionssuch as the maintenance of gastrointestinal integrity and renal bloodflow. In contrast, we have concluded that the inducible form, COX-2, ismainly responsible for the pathological effects of prostaglandins whererapid induction of the enzyme would occur in response to such agents asinflammatory agents, hormones, growth factors, and cytokines. Thus, aselective inhibitor of COX-2 will have similar antiinflammatory,antipyretic and analgesic properties to a conventional non-steroidalantiinflammatory drug, and in addition would inhibit hormone-induceduterine contractions and have potential anti-cancer effects, but willhave a diminished ability to induce some of the mechanism-based sideeffects. In particular, such a compound should have a reduced potentialfor gastrointestinal toxicity, a reduced potential for renal sideeffects, a reduced effect on bleeding times and possibly a lessenedability to induce asthma attacks in aspirin-sensitive asthmaticsubjects.

Furthermore, such a compound will also inhibit prostanoid-induced smoothmuscle contraction by preventing the synthesis of contractileprostanoids and hence may be of use in the treatment of dysmenorrhea,premature labour, asthma and eosinophil related disorders. It will alsobe of use in the treatment of Alzheimer's disease, for decreasing boneloss particularly in postmenopausal women (i.e. treatment ofosteoporosis) and for the treatment of glaucoma.

The potential utilities of selective cyclooxygenase-2 inhibitors arediscussed in the following articles:

1. John Vane, "Towards a better aspirin" in Nature, Vol. 367, pp.215-216, 1994.

2. Bruno Battistini, Regina Botting and Y. S. Bakhle, "COX-1 and COX-2:Toward the Development of More Selective NSAIDs" in Drug News andPerspectives, Vol. 7, pp. 501-512, 1994.

3. David B. Reitz and Karen Seibert, "Selective CyclooxygenaseInhibitors" in Annual Reports in Medicinal Chemistry, James A. Bristol,Editor, Vol. 30, pp. 179-188, 1995.

4. Don E. Griswold and Jerry L. Adams, "Constituative Cyclooxygenase(COX-1) and Inducible Cyclooxygenase (COX-2): Rationale for SelectiveInhibition and Progress to Date" in Medicinal Research Reviews, Vol. 16,pp. 181-206, 1996.

WO 96/10012 (DuPont Merck, Apr. 4, 1996) discloses compounds representedby Formula A as being useful in the treatment of COX-2 mediateddiseases, by virtue of their selective inhibition of COX-2 rather thanCOX-1. We have now discovered that a subset of the compounds representedby A, in which --J--K--L-- is --NCHCH--, X is a bond, R¹ is aromatic andR³ and R⁴ are not both hydrogen show unexpectedly superior selectivityfor the inhibition of COX-2 over COX-1 and/or superior potency ascompared to the closest species disclosed in 96/10012. This subset ofcompounds is the subject of the present invention and is represented byFormula I. ##STR2##

Of the over 175 specific compounds disclosed in WO 96/10012, only 4 ofthem are pyridines, and none of these latter contain a substituent (R³or R⁴ in A) on the pyridine ring.

WO 96/16934 (Searle, Jun. 6, 1996) discloses compounds represented bystructure B as being useful for the treatment of inflammation andrelated disorders. Chemically, these compounds differ from those of thepresent invention in that the central of the three aromatic rings isbenzene rather than pyridine.

SUMMARY OF THE INVENTION

The invention encompasses the novel compound of Formula I as well as amethod of treating COX-2 mediated diseases comprising administration toa patient in need of such treatment of a non-toxic therapeuticallyeffective amount of a compound of Formula I. ##STR3##

The invention also encompasses certain pharmaceutical compositions fortreatment of COX-2 mediated diseases comprising compounds of Formula I.

DETAILED DESCRIPTION OF THE INVENTION

The invention encompasses the novel compound of Formula I as well as amethod of treating COX-2 mediated diseases comprising administration toa patient in need of such treatment of a non-toxic therapeuticallyeffective amount of a compound of Formula I ##STR4## wherein: R¹ isselected from the group consisting of

(a) CH₃,

(b) NH₂,

(c) NHC(O)CF₃,

(d) NHCH₃ ;

Ar is a mono-, di-, or trisubstituted phenyl or pyridinyl (or theN-oxide thereof), wherein the substituents are chosen from the groupconsisting of

(a) hydrogen,

(b) halo,

(c) C₁₋₆ alkoxy,

(d) C₁₋₆ alkylthio,

(e) CN,

(f) C₁₋₆ alkyl,

(g) C₁₋₆ fluoroalkyl,

(h) N₃,

(i) --CO₂ R³,

(j) hydroxy,

(k) --C(R⁴)(R⁵)--OH,

(l) --C₁₋₆ alkyl-CO₂ --R⁶,

(m) C₁₋₆ fluoroalkoxy;

R² is chosen from the group consisting of

(a) halo,

(b) C₁₋₆ alkoxy,

(c) C₁₋₆ alkylthio,

(d) CN,

(e) C₁₋₆ alkyl,

(f) C₁₋₆ fluoroalkyl,

(g) N₃,

(h) --CO₂ R⁷,

(i) hydroxy,

(j) --C(R⁸)(R⁹)--OH,

(k) --C₁₋₆ alkyl-CO₂ --R¹⁰,

(l) C₁₋₆ fluoroalkoxy,

(m) NO₂,

(n) NR¹¹ R¹², and

(o) NHCOR¹³,

R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, are each independantlychosen from the group consisting of

(a) hydrogen, and

(b) C₁₋₆ alkyl,

or R⁴ and R⁵, R⁸ and R⁹ or R ¹¹ and R¹² together with the atom to whichthey are attached form a saturated monocyclic ring of 3, 4, 5, 6 or 7atoms.

Apt alkyl groups include methyl, ethyl, n-propyl, iso-propyl and butyl,pentyl and hexyl groups. A favoured alkyl group is the methyl group. Ingeneral R⁴ and R⁵, R⁸ and R⁹ and R¹¹ and R¹² are not residues of theabove-mentioned monocyclic rings. When `alkyl` is part of a compositeterm such as alkoxy, alkylthio, fluoroalkyl, fluoroalkoxy then the abovemeaning of alkyl refers also to the composite term.

A preferred sub-genus of formula I is that wherein Ar is a mono-, ordisubstituted pyridinyl. Within this sub-genus, the 3-pyridinyl isomers,such as those of formula Ic, are particularly preferred.

When Ar is di-substituted phenyl is particularly apt that one or both ofthe substituents are hydrogen.

Another preferred sub-genus of formula I is that wherein Ar is a mono-or disubstituted phenyl.

When Ar is di-substituted phenyl it is particularly apt that one of thesubstituents is hydrogen or fluorine and the second is hydrogen,fluorine, chlorine, methyl, methoxyl or trifluoromethyl.

Another preferred sub-genus of formula I is that wherein R¹ is CH₃ orNH₂. Generally, CH₃ is preferred for COX-2 specificity and NH₂ ispreferred for potency.

Another preferred sub-genus of formula I is that wherein R² is halo, CH₃or CF₃.

Another preferred sub-genus of formula I is that wherein thesubstituents on Ar are chosen from the group consisting of

(a) hydrogen,

(b) halo,

(c) C₁₋₄ alkoxy,

(d) C₁₋₄ alkylthio,

(e) C₁₋₄ alkyl,

(f) CF₃, and

(g) CN.

In one aspect the invention is directed to compounds of formula I##STR5## wherein: R¹ is selected from the group consisting of

(a) CH₃,

(b) NH₂,

(c) NHC(O)CF₃,

(d) NHCH₃ ;

Ar is a mono-, di-, or trisubstituted pyridinyl (or the N-oxidethereof), wherein the substituents are chosen from the group consistingof

(a) hydrogen,

(b) halo,

(c) C₁₋₆ alkoxy,

(d) C₁₋₆ alkylthio,

(e) CN,

(f) C₁₋₆ alkyl,

(g) C₁₋₆ fluoroalkyl,

(h) N₃,

(i) --CO₂ R³,

(j) hydroxy,

(k) --C(R⁴)(R⁵)--OH,

(l) --C₁₋₆ alkyl-CO₂ --R⁶,

(m) C₁₋₆ fluoroalkoxy;

R² is chosen from the group consisting of

(a) halo,

(b) C₁₋₆ alkoxy,

(c) C₁₋₆ alkylthio,

(d) C₁₋₆ alkyl,

(e) N₃,

(f) --CO₂ H,

(g) hydroxy,

(h) C₁₋₆ fluoroalkoxy,

(i) NO₂,

(j) NR¹¹ R¹², and

(k) NHCOR¹³,

R³, R⁴, R⁵, R⁶, R¹¹, R¹², R¹³, are each independantly chosen from thegroup consisting of

(a) hydrogen, and

(b) C₁₋₆ alkyl,

or R⁴ and R⁵ or R¹¹ and R¹² together with the atom to which they areattached form a saturated monocyclic ring of 3, 4, 5, 6 or 7 atoms.

Within this aspect there is a genus of compounds of formula ##STR6##wherein: R¹ is selected from the group consisting of

(a) CH₃,

(b) NH₂,

R² is chosen from the group consisting of

(a) chloro,

(b) methyl,

and wherein there may be one, two or three groups X independentlyselected from the group consisting of

(a) hydrogen,

(b) halo,

(c) C₁₋₄ alkoxy,

(d) C₁₋₄ alkylthio,

(e) CN,

(f) C₁₋₄ alkyl,

(g) CF3.

Within this genus of compounds of formula Ic ##STR7## there is asub-genes wherein: R¹ is selected from the group consisting of

(a) CH₃,

(b) NH₂,

R² is chloro,

wherein there is one group X independently selected from the groupconsisting of

(a) hydrogen,

(b) F or Cl,

(c) methyl,

(d) ethyl.

Within this genus of compounds of formula Ic ##STR8## there is asub-genes wherein: R¹ is selected from the group consisting of

(a) CH₃,

(b) NH₂,

R² is chloro,

wherein there is one group X independently selected from the groupconsisting of

(a) hydrogen,

(b) F or Cl,

(c) methyl.

Preferred compounds of formula I and Ic include those wherein R² ishalo, especially chloro.

Preferred compounds of formula I and Ic include those wherein Ar is3-pyridinyl and X is hydrogen or C₁₋₃ alkyl, especially hydrogen,p-methyl and p-ethyl.

Illustrating the invention are the following compounds:

3-(4-Methylsulfonyl)phenyl-2-phenyl-5-trifluoromethylpyridine;

2-(3-Chlorophenyl)-3-(4-methylsulfonyl)phenyl-5-trifluoromethyl-pyridine;

2-(4-Chlorophenyl)-3-(4-methylsulfonyl)phenyl-5-trifluoromethyl-pyridine;

2-(4-Fluorophenyl)-3-(4-methylsulfonyl)phenyl-5-trifluoromethyl-pyridine;

3-(4-Methylsulfonyl)phenyl-2-(3-pyridinyl)-5-trifluoromethylpyridine;

5-Methyl-3-(4-methylsulfonyl)phenyl-2-phenylpyridine;

2-(4-Chlorophenyl)-5-methyl-3-(4-methylsulfonyl) phenylpyridine;

5-Methyl-3-(4-methylsulfonyl)phenyl-2-(3-pyridinyl) pyridine;

5-Chloro-2-(4-chlorophenyl)-3-(4-methylsulfonyl) phenylpyridine;

5-Chloro-3-(4-methylsulfonyl)phenyl-2-(2-pyridinyl) pyridine;

5-Chloro-3-(4-methylsulfonyl)phenyl-2-(3-pyridinyl) pyridine;

5-Chloro-3-(4-methylsulfonyl)phenyl-2-(4-pyridinyl) pyridine;

5-Chloro-3-(4-methylsulfonyl)phenyl-2-(2-methyl-5-pyridinyl)pyridine;

2-(4-Chlorophenyl)-3-(4-methylsulfonyl)phenylpyridinyl-5-carboxylic acidmethyl ester;

2-(4-Chlorophenyl)-3-(4-methylsulfonyl)phenylpyridinyl-5-carboxylicacid;

5-Cyano-2-(4-chlorophenyl)-3-(4-methylsulfonyl) phenylpyridine;

5-Chloro-3-(4-methylsulfonyl)phenyl-2-(3-pyridyl)pyridinehydromethanesulfonate;

5-Chloro-3-(4-methylsulfonyl)phenyl-2-(3-pyridyl)pyridine hydrochloride;

5-Chloro-3-(4-methylsulfonyl)phenyl-2-(2-methyl-5-pyridinyl)pyridineHydrochloride;

5-Chloro-3-(4-methylsulfonyl)phenyl-2-(2-ethyl-5-pyridinyl)pyridine; and

5-Chloro-3-(4-methylsulfonyl)phenyl-2-(2-ethyl-5-pyridinyl)pyridinehydromethanesulfonate.

Preferred compounds of formula I and Ic include those wherein R¹ ismethyl or NH₂, especially methyl.

In another aspect the invention also encompasses a pharmaceuticalcomposition for treating an inflammatory disease susceptable totreatment with an non-steroidal anti-inflammatory agent comprising:

a non-toxic therapeutically effective amount of a compound of formula Iand a pharmaceutically acceptable carrier.

In another aspect the invention also encompasses a pharmaceuticalcomposition for treating cyclooxygenase mediated diseases advantageouslytreated by an active agent that selectively inhibits COX-2 in preferenceto COX-1 comprising:

a non-toxic therapeutically effective amount of a compound of formula Iand a pharmaceutically acceptable carrier.

In another aspect the invention also encompasses a method of treating aninflammatory disease susceptable to treatment with an non-steroidalanti-inflammatory agent comprising:

administration to a patient in need of such treatment of a non-toxictherapeutically effective amount of a compound of formula I and apharmaceutically acceptable carrier.

In another aspect the invention also encompasses a method of treatingcyclooxygenase mediated diseases advantageously treated by an activeagent that selectively inhibits COX-2 in preference to COX-1 comprising:

administration to a patient in need of such treatment of a non-toxictherapeutically effective amount of a compound of formula I.

In another aspect the invention also encompasses the use of a compoundof formula I or a pharmaceutical composition in the manufacture of amedicament for the treatment of an inflammatory disease susceptable totreatment with an a non-steroidal anti-inflammatory agent.

The invention is illustrated by Example 1 to 56.

The following abbreviations have the indicated meanings:

AA=arachidonic acid

Ac=acetyl

AIBN=2.2⁻ -azobisisobutyronitrile

BHT=butylated hydroxytoluene

Bn=benzyl

CSA=camphor sulfonic acid (racemic)

dba=dibenzylideneacetone

DMAP=4-(dimethylamino)pyridine

DMF=N,N-dimethylformamide

DMSO=dimethyl sulfoxide

EDTA=ethylenediaminetetraacetic acid

ESA=ethane sulfonic acid

Et₃ N=triethylamine

HBSS=Hanks balanced salt solution

HEPES=N-[2-Hydroxyethyl]piperazine-N¹ -[2-ethanesulfonic acid]

HWB=human whole blood

KHMDS=potassium hexamethyldisilazane

LDA=lithium diisopropylamide

LPS=lipopolysaccharide

mCPBA=m-chloroperbenzoic acid

MMPP=magnesium monoperoxyphthalate

Ms=methanesulfonyl=mesyl

Ms0=methanesulfonate=mesylate

MTBE=methyl tert-butyl ether

NBS=N-bromosuccinimide

NCS=N-chlorosuccinimide

NIS=N-iodosuccinimide

NMO=N-methylmorpholine-N-oxide

NMP=N-methylpyrrolidone

NSAID=non-steroidal anti-inflammatory drug

oxone®=2KHSO₅.KHSO₄.K₂ SO₄

PCC=pyridinium chlorochromate

PDC=pyridinium dichromate

PEG=polyethyleneglycol

Ph=phenyl

pyr=pyridinyl

r.t.=room temperature

rac.=racemic

Tf=trifluoromethanesulfonyl=triflyl

TfO=trifluoromethanesulfonate=triflate

THF=tetrahydrofuran

TLC=thin layer chromatography

Ts=p-toluenesulfonyl=tosyl

TsO=p-toluenesulfonate=tosylate

Tz=1H (or 2H)-tetrazol-5-yl

SO₂ Me=methyl sulfone

SO₂ NH₂ =sulfonamide

Alkyl group abbreviations

Me=methyl

Et=ethyl

n-Pr=normal propyl

i-Pr isopropyl

n-Bu=normal butyl

i-Bu=isobutyl

s-Bu=secondary butyl

t-Bu=tertiary butyl

c-Pr=cyclopropyl

c-Bu=cyclobutyl

c-Pen=cyclopentyl

c-Hex=cyclohexyl

Dose Abbreviations

bid=bis in die=twice daily

qid=quater in die=four times a day

tid=ter in die=three times a day

For purposes of this specification alkyl is defined to include linear,branched and cyclic stuctures, with the indicated number of carbonatoms. Examples of alkyl are methyl, ethyl, propyl, s- and t-butyl,butyl, pentyl, hexyl, 1,1-dimethylethyl, cyclopropyl, cyclobutyl,cyclohexylmethyl and the like. Similarly, alkoxy and alkylthio meanlinear, branched and cyclic stuctures, with the indicated number ofcarbon atoms.

For purposes of this specification fluoroalkyl means alkyl groups of theindicated number of carbon atoms in which one hydrogen or more isreplaced by fluorine. Examples are --CF₃, --CH₂ CH₂ F, --CH₂ CF₃,c-Pr-F₅, c-Hex-F₁₁ and the like. Similarly, fluoroalkoxy means linear,branched and cyclic stuctures, with the indicated number of carbonatoms.

For purposes of this specification, in situations in which a term occurstwo or more times, the definition of the term in each occurrence isindependent of the definition in each additional occurrence.

For purposes of this specification halo means F, Cl, Br, or I.

In another embodiment, the invention encompasses pharmaceuticalcompositions for inhibiting COX-2 and for treating COX-2 mediateddiseases as disclosed herein comprising a pharmaceutically acceptablecarrier and non-toxic therapeutically effective amount of a compound offormula I as described above.

In yet another embodiment, the invention encompasses a method ofinhibiting cyclooxygenase and treating cyclooxygenase mediated diseases,advantageously treated by an active agent that selectively inhibitsCOX-2 in preference to COX-1 as disclosed herein comprising:

administration to a patient in need of such treatment of a non-toxictherapeutically effective amount of a compound of Formula I as disclosedherein.

Optical Isomers--Diastereomers--Geometric Isomers

Some of the compounds described herein contain one or more asymmetriccentres and may thus give rise to diastereomers and optical isomers. Thepresent invention is meant to comprehend such possible diastereomers aswell as their racemic and resolved, enantiomerically pure forms andpharmaceutically acceptable salts thereof.

Some of the compounds described herein contain olefinic double bonds,and unless specified otherwise, are meant to include both E and Zgeometric isomers.

Salts

The pharmaceutical compositions of the present invention comprise acompound of Formula I as an active ingredient or a pharmaceuticallyacceptable salt, thereof, and may also contain a pharmaceuticallyacceptable carrier and optionally other therapeutic ingredients. Theterm "pharmaceutically acceptable salts" refers to salts prepared frompharmaceutically acceptable non-toxic bases including inorganic basesand organic bases. Salts derived from inorganic bases include aluminum,ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganicsalts, manganous, potassium, sodium, zinc, and the like. Particularlypreferred are the ammonium, calcium, magnesium, potassium, and sodiumsalts. Salts derived from pharmaceutically acceptable organic non-toxicbases include salts of primary, secondary, and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines, and basic ion exchange resins, such as arginine, betaine,caffeine, choline, N,N⁻ -dibenzylethylenediamine, diethylamine,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,ethylenediamine, N-ethylmorpholine, N-ethylpiperidine,N-methylglucamine, glucamine, glucosamine, histidine, hydrabamine,N-(2-hydroxyethyl)piperidine, N-(2-hydroxyethyl)pyrrolidine,isopropylamine, lysine, methylglucamine, morpholine, piperazine,piperidine, polyamine resins, procaine, purines, theobromine,triethylamine, trimethylamine, tripropylamine, tromethamine, and thelike.

When the compound of the present invention is basic, salts may beprepared from pharmaceutically acceptable non-toxic acids, includinginorganic and organic acids. Such acids include acetic, adipic,aspartic, 1,5-naphthalenedisulfonic, benzenesulfonic, benzoic,camphorsulfonic, citric, 1,2-ethanedisulfonic, ethanesulfonic,ethylenediaminetetraacetic, fumaric, glucoheptonic, gluconic, glutamic,hydriodic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic,mandelic, methanesulfonic, mucic, 2-naphthalenesulfonic, nitric, oxalic,pamoic, pantothenic, phosphoric, pivalic, propionic, salicylic, stearic,succinic, sulfuric, tartaric, p-toluenesulfonic acid, undecanoic,10-undecenoic, and the like. Particularly preferred are citric,hydrobromic, hydrochloric, maleic, methanesulfonic, phosphoric, sulfuricand tartaric acids.

It will be understood that in the discussion of methods of treatmentwhich follows, references to the compounds of Formula I are meant toalso include the pharmaceutically acceptable salts.

Utilities

The Compound of Formula I is useful for the relief of pain, fever andinflammation of a variety of conditions including rheumatic fever,symptoms associated with influenza or other viral infections, commoncold, low back and neck pain, dysmenorrhea, headache, toothache, sprainsand strains, myositis, neuralgia, synovitis, arthritis, includingrheumatoid arthritis, degenerative joint diseases (osteoarthritis), goutand ankylosing spondylitis, bursitis, burns, injuries, followingsurgical and dental procedures. In addition, such a compound may inhibitcellular neoplastic transformations and metastic tumour growth and hencecan be used in the treatment of cancer. Compound 1 may also be of use inthe treatment and/or prevention of cyclooxygenase-mediated proliferativedisorders such as may occur in diabetic retinopathy and tumourangiogenesis.

Compound I will also inhibit prostanoid-induced smooth musclecontraction by preventing the synthesis of contractile prostanoids andhence may be of use in the treatment of dysmenorrhea, premature labour,asthma and eosinophil related disorders. It will also be of use in thetreatment of Alzheimer's disease, for decreasing bone loss particularlyin postmenopausal women (i.e. treatment of osteoporosis) and fortreatment of glaucoma.

By virtue of its high inhibitory activity against COX-2 and/or itsspecificity for inhibiting COX-2 over COX-1, compound I will proveuseful as an alternative to conventional NSAID'S, particularly wheresuch non-steroidal antiinflammatory drugs may be contra-indicated suchas in patients with peptic ulcers, gastritis, regional enteritis,ulcerative colitis, diverticulitis or with a recurrent history ofgastrointestinal lesions; GI bleeding, coagulation disorders includinganaemia such as hypoprothrombinemia, haemophilia or other bleedingproblems; kidney disease; those prior to surgery or takinganticoagulants.

Pharmaceutical Compositions

For the treatment of any of these cyclooxygenase mediated diseasescompound I may be administered orally, topically, parenterally, byinhalation spray or rectally in dosage unit formulations containingconventional non-toxic pharmaceutically acceptable carriers, adjuvantsand vehicles. The term parenteral as used herein includes subcutaneousinjections, intravenous, intramuscular, intrasternal injection orinfusion techniques. In addition to the treatment of warm-bloodedanimals such as mice, rats, horses, cattle, sheep, dogs, cats, etc., thecompound of the invention is effective in the treatment of humans. Thecompounds of the instant invention are particularly well suited forhorses.

As indicated above, pharmaceutical compositions for treating COX-2mediated diseases as defined may optionally include one or moreingredients as listed above.

The pharmaceutical compositions containing the active ingredient may bein a form suitable for oral use, for example, as tablets, troches,lozenges, aqueous or oily suspensions, dispersible powders or granules,emulsions, hard or soft capsules, or syrups or elixirs. Compositionsintended for oral use may be prepared according to any method known tothe art for the manufacture of pharmaceutical compositions and suchcompositions may contain one or more agents selected from the groupconsisting of sweetening agents, flavouring agents, colouring agents andpreserving agents in order to provide pharmaceutically elegant andpalatable preparations. Tablets contain the active ingredient inadmixture with non-toxic pharmaceutically acceptable excipients whichare suitable for the manufacture of tablets. These excipients may be forexample, inert diluents, such as calcium carbonate, sodium carbonate,lactose, calcium phosphate or sodium phosphate; granulating anddisintegrating agents, for example, corn starch, or alginic acid;binding agents, for example starch, gelatin or acacia, and lubricatingagents, for example, magnesium stearate, stearic acid or talc. Thetablets may be uncoated or they may be coated by known techniques todelay disintegration and absorption in the gastrointestinal tract andthereby provide a sustained action over a longer period. For example, atime delay material such as glyceryl monostearate or glyceryl distearatemay be employed. They may also be coated by the technique described inthe U.S. Pat. Nos. 4,256,108; 4,166,452; and 4,265,874 to form osmotictherapeutic tablets for control release.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredients is mixed with water ormiscible solvents such as propylene glycol, PEGs and ethanol, or an oilmedium, for example peanut oil, liquid paraffin, or olive oil.

Aqueous suspensions contain the active material in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethycellulose,sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents may be a naturally-occurring phosphatide,for example lecithin, or condensation products of an alkylene oxide withfatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions may also contain one or more preservatives, forexample ethyl, or n-propyl, p-hydroxybenzoate, benzyl alcohol, one ormore colouring agents, one or more flavouring agents, and one or moresweetening agents, such as sucrose, saccharin or aspartame.

Oily suspensions may be formulated by suspending the active ingredientin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set forthabove, and flavouring agents may be added to provide a palatable oralpreparation. These compositions may be preserved by the addition of ananti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavouring and colouringagents, may also be present.

The pharmaceutical compositions of the invention may also be in the formof an oil-in-water emulsions. The oily phase may be a vegetable oil, forexample olive oil or arachis oil, or a mineral oil, for example liquidparaffin or mixtures of these. Suitable emulsifying agents may benaturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitolanhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions may also containsweetening and flavouring agents.

Syrups and elixirs may be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol or sucrose. Such formulations mayalso contain a demulcent, a preservative and flavouring and colouringagents. The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleagenous suspension. This suspension may beformulated according to the known art using those suitable dispersing orwetting agents and suspending agents which have been mentioned above.The sterile injectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parenterally-acceptable diluent orsolvent, for example as a solution in 1,3-butane diol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution and isotonic sodium chloride solution. Cosolvents suchas ethanol, propylene glycol or polyethylene glycols may also be used.In addition, sterile, fixed oils are conventionally employed as asolvent or suspending medium. For this purpose any bland fixed oil maybe employed including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation ofinjectables.

Compound of Formula I may also be administered in the form of asuppositories for rectal administration of the drug. These compositionscan be prepared by mixing the drug with a suitable non-irritatingexcipient which is solid at ordinary temperatures but liquid at therectal temperature and will therefore melt in the rectum to release thedrug. Such materials are cocoa butter and polyethylene glycols.

For topical use, creams, ointments, gels, solutions or suspensions,etc., containing the compound of Formula I are employed. (For purposesof this application, topical application shall include mouth washes andgargles.) Topical formulations may generally be comprised of apharmaceutical carrier, cosolvent, emulsifier, penetration enhancer,preservative system, and emollient.

Dose Ranges

Dosage levels of the order of from about 0.01 mg to about 140 mg/kg ofbody weight per day are useful in the treatment of the above-indicatedconditions, or alternatively about 0.5 mg to about 7 g per patient perday. For example, inflammation may be effectively treated by theadministration of from about 0.01 to 50 mg of the compound per kilogramof body weight per day, or alternatively about 0.5 mg to about 3.5 g perpatient per day.

The amount of active ingredient that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. For example, aformulation intended for the oral administration of humans may containfrom 0.5 mg to 5 g of active agent compounded with an appropriate andconvenient amount of carrier material which may vary from about 5 toabout 95 percent of the total composition. Dosage unit forms willgenerally contain between from about 1 mg to about 500 mg of an activeingredient, typically 25 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500mg, 600 mg, 800 mg, or 1000 mg.

It will be understood, however, that the specific dose level for anyparticular patient will depend upon a variety of factors including theage, body weight, general health, sex, diet, time of administration,route of administration, rate of excretion, drug combination and theseverity of the particular disease undergoing therapy.

Combinations with Other Drugs

Similarly, compound of Formula I, will be useful as a partial orcomplete substitute for conventional NSAID'S in preparations whereinthey are presently co-administered with other agents or ingredients.Thus in further aspects, the invention encompasses pharmaceuticalcompositions for treating COX-2 mediated diseases as defined abovecomprising a non-toxic therapeutically effective amount of the compoundof Formula I as defined above and one or more ingredients such asanother pain reliever including acetaminophen or phenacetin; apotentiator including caffeine; an H₂ -antagonist, aluminum or magnesiumhydroxide, simethicone, a decongestant including phenylephrine,phenylpropanolamine, pseudophedrine, oxymetazoline, ephinephrine,naphazoline, xylometazoline, propylhexedrine, or levodesoxyephedrine; anantiitussive including codeine, hydrocodone, caramiphen, carbetapentane,or dextramethorphan; a prostaglandin including misoprostol, enprostil,rioprostil, ornoprostol or rosaprostol: a diuretic; a sedating ornon-sedating antihistamine. In addition the invention encompasses amethod of treating cyclooxygenase mediated diseases comprising:administration to a patient in need of such treatment a non-toxictherapeutically effective amount of the compound of Formula I,optionally co-administered with one or more of such ingredients aslisted immediately above.

Methods of Synthesis

The compounds of Formula I of the present invention can be preparedaccording to the synthetic routes outlined in Schemes 1 to 2 and byfollowing the methods described therein.

SCHEME 1

The pyridines of Formula Ia and Ib may be prepared in a multi-stepsequence from the requisite 2-aminopyridine II. Initial bromination ofII with bromine in acetic acid provides the bromide III. Apalladium-catalyzed coupling of III with 4-(methylthio)phenyl-boronicacid in the presence of a suitable base, such as sodium carbonate,provides the sulfide IV which can be oxidized using one of severaloxidants, such as MMPP, oxone ®, or OsO₄ /NMO to the correspondingsulfone V. The amino pyridine V can be converted to the halide VI viaone of several methods. For example, treatment of V with sodium nitritein the presence of HCl and bromine provides the bromide VI (X=Br).Alternatively, treatment of V with sodium nitrite and HCl followed byreaction with POCl₃ affords the corresponding chloride VI (X=Cl). Asecond palladium-catalyzed coupling of VI with an appropriatelysubstituted metalated aromatic, such as an aryl boronic acid or an arylstannane, provides the pyridine of Formula Ia. Suitable modification ofthe R² substituent in Ia provides additional examples of Ia. For examplewhen R² =Me, oxidation with an oxidant such as KMnO₄ provides thecorresponding acid (Ia R² =CO₂ H) which can then be converted to themethyl ester (Ia R² =CO₂ Me), using a reagent such as diazomethane.Alternatively, treatment of the acid with chlorosulfonylisocyanate andDMF provides the nitrile (Ia R² =CN). The pyridine methyl sulfones Iacan be converted to the corresponding pyridine sulfonamides Ib by usingprocedures described in the literature (Huang et. al. Tetrahedron Lett.1994, 39, 7201). ##STR9##

SCHEME 2

The 2-halopyridines VI of Scheme 1 can also be prepared in a multi-stepprocess from the appropriate 2-hydroxypyridines VII. First, treatment ofVII with bromine in acetic acid provides the bromide VIII. Subsequentreaction of VIII with benzyl bromide in the presence of a base such assilver carbonate yields the benzyl ether IX which can be converted tothe sulfone X via a sequence of reactions similar to those described forthe conversion of bromide III to V in Scheme 1. The benzyl protectinggroup can be removed by treatment of IX with an acid such astrifluoroacetic acid to afford the hydroxypyridine X. Heating X withPOBr₃ or POCl₃ provides the corresponding 2-halopyridines VI (X=Br, Cl)of Scheme 1. ##STR10##

REPRESENTATIVE COMPOUNDS

Tables 1 and 2 illustrate compounds of formula Ia and Ib, which arerepresentative of the present invention.

                  TABLE 1                                                         ______________________________________                                                                      Ia                                               ##STR11##                                                                    Ex.          R.sup.2      Ar                                                  ______________________________________                                         1           CF.sub.3     Ph                                                   2           CF.sub.3     3-ClC.sub.6 H.sub.4                                  3           CF.sub.3     4-ClC.sub.6 H.sub.4                                  4           CF.sub.3     4-FC.sub.6 H.sub.4                                   5           CF.sub.3     2-(CMe.sub.2 OH)C.sub.6 H.sub.4                      6           CF.sub.3     3-(CMe.sub.2 OH)C.sub.6 H.sub.4                      7           CF.sub.3     3-pyr                                                8           CF.sub.3     5-(2-Me)pyr                                          9           CF.sub.3     5-(3-Br)pyr                                         10           CF.sub.3     5-(3-Cl)pyr                                         11           CF.sub.3     5-(2-OMe)pyr                                        12           CF.sub.3     2-(5-Br)pyr                                         13           Me           Ph                                                  14           Me           4-ClC.sub.6 H.sub.4                                 15           Me           3-pyr                                               16           Cl           Ph                                                  17           Cl           4-ClC.sub.6 H.sub.4                                 18           Cl           2-(CMe.sub.2 OH)C.sub.6 H.sub.4                     19           Cl           3-(CMe.sub.2 OH)C.sub.6 H.sub.4                     20           Cl           2-pyr                                               21           Cl           3-pyr                                               22           Cl           4-pyr                                               23           Cl           5-(2-Me)pyr                                         24           Cl           5-(3-Br)pyr                                         25           Cl           5-(3-Cl)pyr                                         26           Cl           5-(2-OMe)pyr                                        27           Cl           2-(5-Br)pyr                                         28           F            Ph                                                  29           F            3-pyr                                               30           F            5-(2-Me)pyr                                         31           Br           Ph                                                  32           Br           3-pyr                                               33           Br           5-(2-Me)pyr                                         34           NO.sub.2     Ph                                                  35           NO.sub.2     3-pyr                                               36           NO.sub.2     5-(2-Me)pyr                                         37           OMe          Ph                                                  38           OMe          3-pyr                                               39           OMe          5-(2-Me)pyr                                         40           NHCOMe       Ph                                                  41           NHCOMe       3-pyr                                               42           NHCOMe       5-(2-Me)pyr                                         43           CO.sub.2 Me  4-ClC.sub.6 H.sub.4                                 44           CO.sub.2 H   4-ClC.sub.6 H.sub.4                                 45           CN           4-ClC.sub.6 H.sub.4                                 46           Cl           3-pyr.MeSO.sub.3 H                                  47           Cl           3-pyr.HCl                                           57           Cl           3-pyr.CSA                                           58           Cl           3-pyr.ESA                                           59           Cl           5-(2-Me)pyr.HCl                                     60           Cl           5-(2-CH.sub.2 OH)pyr                                61           Cl           5-(2-CO.sub.2 H)pyr                                 62           Cl           5-(2-Me)pyr-N-oxide                                 63           Cl           5-(3-Me)pyr                                         64           Cl           3-(4-Me)pyr                                         65           Cl           3-(2-Me)pyr                                         66           Cl           3-(2-Et)pyr                                         67           Cl           3-(2-c-Pr)pyr                                       68           Me           3-pyr.HCl                                           69           CN           3-pyr                                               70           CN           5-(2-Me)pyr                                         71           Cl           5-(2-Et)pyr                                         72           Cl           5-(2-Et)pyr-MeSO.sub.3 H                            73           Cl           5-(2-c-Pr)pyr                                       74           Cl           3-(2,6-Me.sub.2)pyr                                 ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                                                      Ib                                               ##STR12##                                                                    Ex.           R.sup.2     Ar                                                  ______________________________________                                        48            CF.sub.3    Ph                                                  49            CF.sub.3    4-ClC.sub.6 H.sub.4                                 50            CF.sub.3    4-FC.sub.6 H.sub.4                                  51            CF.sub.3    3-pyr                                               52            Me          Ph                                                  53            Me          4-ClC.sub.6 H.sub.4                                 54            Cl          3-pyr                                               55            Cl          5-(2-Me)pyr                                         56            CN          4-ClC.sub.6 H.sub.4                                 ______________________________________                                    

ASSAYS FOR DETERMINING BIOLOGICAL ACTIVITY

The compound of Formula I can be tested using the following assays todetermine their COX-2 inhibiting activity.

INHIBITION OF CYCLOOXYGENASE ACTIVITY

Compounds are tested as inhibitors of cyclooxygenase activity in wholecell cyclooxygenase assays. Both of these assays measure prostaglandinE₂ synthesis in response to AA, using a radioimmunoassay. Cells used forthese assays are human osteosarcoma 143 cells (which specificallyexpress COX-2) and human U-937 cells (which specifically express COX-1).In these assays, 100% activity is defined as the difference betweenprostaglandin E₂ synthesis in the absence and presence of arachidonate.

Whole Cell Assays

For cyclooxygenase assays, osteosarcoma cells are cultured in 1 mL ofmedia in 24-well multidishes (Nunclon) until confluent (1-2×10⁵cells/well). U-937 cells are grown in spinner flasks and resuspended toa final density of 1.5×10⁶ cells/mL in 24-well multidishes (Nunclon).Following washing and resuspension of osteosarcoma and U-937 cells in 1mL of HBSS, 1 μL of a DMSO solution of test compound or DMSO vehicle isadded, and samples gently mixed. All assays are performed in triplicate.Samples are then incubated for 5 or 15 minutes at 37° C., prior to theaddition of AA. AA (peroxide-free, Cayman Chemical) is prepared as a 10mM stock solution in ethanol and further diluted 10-fold in HBSS. Analiquot of 10 μL of this diluted solution is added to the cells to givea final AA concentration of 10 μM. Control samples are incubated withethanol vehicle instead of AA. Samples are again gently mixed andincubated for a further 10 min. at 37° C. For osteosarcoma cells,reactions are then stopped by the addition of 100 μL of 1 N HCl, withmixing and by the rapid removal of the solution from cell monolayers.For U-937 cells, reactions are stopped by the addition of 100 μL of 1 NHCl, with mixing. Samples are then neutralized by the addition of 100 μLof 1 N NaOH and PGE₂ levels measured by radioimmunoassay.

Whole Cell Assays for COX-2 and COX-1 using CHO Transfected Cell Lines

Chinese hamster ovary (CHO) cell lines which have been stablytransfected with an eukaryotic expression vector pCDNAIII containingeither the human COX-1 or COX-2 cDNA's are used for the assay. Thesecell lines are referred to as CHO [hCOX-1] and CHO [hCOX-2],respectively. For cyclooxygenase assays, CHO[hCOX-1] cells fromsuspension cultures and CHO[hCOX-2] cells prepared by trypsinization ofadherent cultures are harvested by centrifugation (300×g, 10 min) andwashed once in HBSS containing 15 mM HEPES, pH 7.4, and resuspended inHBSS, 15 mM HEPES, pH 7.4, at a cell concentration of 1.5×10⁶ cells/ml.Drugs to be tested are dissolved in DMSO to 66.7-fold the highest testdrug concentration. Compounds are typically tested at 8 concentrationsin duplicate using serial 3-fold serial dilutions in DMSO of the highestdrug concentration. Cells (0.3×10⁶ cells in 200 μl) are preincubatedwith 3 μl of the test drug or DMSO vehicle for 15 min at 37° C. Workingsolutions of peroxide-free AA (5.5 μM and 110 μM AA for the CHO [hCOX-1]and CHO [COX-2] assays, respectively) are prepared by a 10-fold dilutionof a concentrated AA solution in ethanol into HBSS containing 15 mMHEPES, pH 7.4. Cells are then challenged in the presence or absence ofdrug with the AA/HBSS solution to yield a final concentration of 0.5 μMAA in the CHO[hCOX-1] assay and a final concentration of 10 μM AA in theCHO[hCOX-2] assay. The reaction is terminated by the addition of 10 μl 1N HCl followed by neutralization with 20 μl of 0.5 N NaOH. The samplesare centrifuged at 300×g at 4° C. for 10 min, and an aliquot of theclarified supernatant is appropriately diluted for the determination ofPGE₂ levels using an enzyme-linked immunoassay for PGE₂ (Correlate PGE₂enzyme immunoassay kit, Assay Designs, Inc.). Cyclooxygenase activity inthe absence of test compounds is determined as the difference in PGE₂levels of cells challenged with AA versus the PGE₂ levels in cellsmock-challenged with ethanol vehicle. Inhibition of PGE₂ synthesis bytest compounds is calculated as a percentage of the activity in thepresence of drug versus the activity in the positive control samples.

Assay of COX-1 Activity from U937 Cell Microsomes

U 937 cells are pelleted by centrifugation at 500×g for 5 min and washedonce with phosphate-buffered saline and repelleted. Cells areresuspended in homogenization buffer consisting of 0.1 M Tris-HCl, pH7.4, 10 mM EDTA, 2 μg/ml leupeptin, 2 μg/ml soybean trypsin inhibitor, 2μg/ml aprotinin and 1 mM phenyl methyl sulfonyl fluoride. The cellsuspension is sonicated 4 times for 10 sec and is centrifuged at10,000×g for 10 min at 4° C. The supernatant is centrifuged at 100,000×gfor 1 hr at 4° C. The 100,000×g microsomal pellet is resuspended in 0.1M Tris-HCl, pH 7.4, 10 mM EDTA to approximately 7 mg protein/ml andstored at -80° C.

Microsomal preparations are thawed immediately prior to use, subjectedto a brief sonication, and then diluted to a protein concentration of125 μg/ml in 0.1 M Tris-HCl buffer, pH 7.4 containing 10 mM EDTA, 0.5 mMphenol, 1 mM reduced glutathione and 1 μM hematin. Assays are performedin duplicate in a final volume of 250 μl. Initially, 5 μl of DMSOvehicle or drug in DMSO are added to 20 μl of 0.1 M Tris-HCl buffer, pH7.4 containing 10 mM EDTA in wells of a 96-deepwell polypropylene titreplate. 200 μl of the microsomal preparation are then added andpre-incubated for 15 min at room temperature before addition of 25 μl of1 M arachidonic acid in 0.1 M Tris-HCl and 10 mM EDTA, pH 7.4. Samplesare incubated for 40 min at room temperature and the reaction is stoppedby the addition of 25 μl of 1 N HCl. Samples are neutralized with 25 μlof 1 N NaOH prior to quantitation of PGE₂ content by radioimmunoassay(Dupont-NEN or Amersham assay kits). Cyclooxygenase activity is definedas the difference between PGE₂ levels in samples incubated in thepresence of arachidonic acid and ethanol vehicle.

Assay of the Activity of Purified Human COX-2

The enzyme activity is measured using a chromogenic assay based on theoxidation of N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) during thereduction of PGG₂ to PGH₂ by COX-2 (Copeland et al. (1994) Proc. Natl.Acad. Sci. 91, 11202-11206).

Recombinant human COX-2 is purified from Sf9 cells as previouslydescribed (Percival et al (1994) Arch. Biochem. Biophys. 15, 111-118).The assay mixture (180 μL) contains 100 mM sodium phosphate, pH 6.5, 2mM genapol X-100, 1 μM hematin, 1 mg/ml gelatin, 80-100 units ofpurified enzyme (One unit of enzyme is defined as the amount of enzymerequired to produce an O.D. change of 0.001/min at 610 nm) and 4 μL ofthe test compound in DMSO. The mixture is pre-incubated at roomtemperature (22° C.) for 15 minutes prior to initiation of the enzymaticreaction by the addition of 20 μL of a sonicated solution of 1 mM AA and1 mM TMPD in assay buffer (without enzyme or hematin). The enzymaticactivity is measured by estimation of the initial velocity of TMPDoxidation over the first 36 sec of the reaction. A non-specific rate ofoxidation is observed in the absence of enzyme (0.007-0.010 O.D./min)and is subtracted before the calculation of the % inhibition. IC₅₀values are derived from 4-parameter least squares non-linear regressionanalysis of the log-dose vs % inhibition plot.

HUMAN WHOLE BLOOD ASSAY

Rationale

Human whole blood provides a protein and cell-rich milieu appropriatefor the study of biochemical efficacy of anti-inflammatory compoundssuch as selective COX-2 inhibitors. Studies have shown that normal humanblood does not contain the COX-2 enzyme. This is consistent with theobservation that COX-2 inhibitors have no effect on PGE₂ production innormal blood. These inhibitors are active only after incubation of humanwhole blood with LPS, which induces COX-2. This assay can be used toevaluate the inhibitory effect of selective COX-2 inhibitors on PGE₂production. As well, platelets in whole blood contain a large amount ofthe COX-1 enzyme. Immediately following blood clotting, platelets areactivated through a thrombin-mediated mechanism. This reaction resultsin the production of thromboxane B₂ (TxB₂) via activation of COX-1.Thus, the effect of test compounds on TxB₂ levels following bloodclotting can be examined and used as an index for COX-1 activity.Therefore, the degree of selectivity by the test compound can bedetermined by measuring the levels of PGE₂ after LPS induction (COX-2)and TxB₂ following blood clotting (COX-1) in the same assay.

METHOD

Step A: COX-2 (LPS-induced PGE₂ Production)

Fresh blood is collected in heparinized tubes by venipuncture from bothmale and female volunteers. The subjects have no apparent inflammatoryconditions and have not taken any NSAIDs for at least 7 days prior toblood collection. Plasma is immediately obtained from a 2 mL bloodaliquot to use as blank (basal levels of PGE₂). The remaining blood isincubated with LPS (100 μg/ml final concentration, Sigma Chem, #L-2630from E. coli; diluted in 0.1% BSA (Phosphate buffered saline) for 5minutes at room temperature. Five hundred μL aliquots of blood areincubated with either 2 μL of vehicle (DMSO) or 2 μL of a test compoundat final concentrations varying from 10 nM to 30 μM for 24 hours at 37°C. At the end of the incubation, the blood is centrifuged at 12,000×gfor 5 minutes to obtain plasma. A 100 μL aliquot of plasma is mixed with400 μL of methanol for protein precipitation. The supernatant isobtained and is assayed for PGE₂ using a radioimmunoassay kit (Amersham,RPA#530) after conversion of PGE₂ to its methyl oximate derivativeaccording to the manufacturer's procedure.

Step B: COX-1 (Clotting-induced TxB₂ Production)

Fresh blood is collected into vacutainers containing no anticoagulants.Aliquots of 500 μL are immediately transferred to siliconizedmicrocentrifuge tubes preloaded with 2 μL of either DMSO or a testcompound at final concentrations varying from 10 nM to 30 μM. The tubesare vortexed and incubated at 37° C. for 1 hour to allow blood to clot.At the end of incubation, serum is obtained by centrifugation (12,000×gfor 5 min.). A 100 μL aliquot of serum is mixed with 400 μL of methanolfor protein precipitation. The supernatant is obtained and is assayedfor TxB₂ using a enzyme immunoassay kit (Cayman, #519031) according tothe manufacturer's instruction.

RAT PAW EDEMA ASSAY

Protocol

Male Sprague-Dawley rats (150-200 g) are fasted overnight and are given,po, either vehicle (1% methocel or 5% Tween 80) or a test compound. Onehr later, a line is drawn using a permanent marker at the level abovethe ankle in one hind paw to define the area of the paw to be monitored.The paw volume (V₀) is measured using a plethysmometer (Ugo-Basile,Italy) based on the principle of water displacement. The animals arethen injected subplantarly with 50 μl of 1% carrageenan solution insaline (FMC Corp, Maine) into the paw using an insulin syringe with a25-gauge needle (i.e. 500 μg carrageenan per paw). Three hr later, thepaw volume (V₃) is measured and the increases in paw volume (V₃ -V_(O))are calculated. The animals are sacrificed by CO₂ asphyxiation and theabsence or presence of stomach lesions scored. Data is compared with thevehicle-control values and percent inhibition calculated. All treatmentgroups are coded to eliminate observer bias.

NSAID-INDUCED GASTROPATHY IN RATS

Rationale

The major side effect of conventional NSAIDs is their ability to producegastric lesions in man. This action is believed to be caused byinhibition of COX-1 in the gastrointestinal tract. Rats are particularlysensitive to the actions of NSAIDs. In fact, rat models have been usedcommonly in the past to evaluate the gastrointestinal side effects ofcurrent conventional NSAIDs. In the present assay, NSAID-inducedgastrointestinal damage is observed by measuring fecal ⁵¹ Cr excretionafter systemic injection of ⁵¹ Cr-labeled red blood cells. Fecal ⁵¹ Crexcretion is a well-established and sensitive technique to detectgastrointestinal integrity in animals and man.

Methods

Male Sprague Dawley rats (150-200 g) are administered orally a testcompound either once (acute dosing) or b.i.d. for 5 days (chronicdosing). Immediately after the administration of the last dose, the ratsare injected via a tail vein with 0.5 mL of ⁵¹ Cr-labeled red bloodcells from a donor rat. The animals are placed individually inmetabolism cages with food and water ad lib. Feces are collected for a48 h period and ⁵¹ Cr fecal excretion is calculated as a percent oftotal injected dose. ⁵¹ Cr-labeled red blood cells are prepared usingthe following procedures. Ten mL of blood is collected in heparinizedtubes via the vena cava from a donor rat. Plasma is removed bycentrifugation and replenished with equal volume of HBSS. The red bloodcells are incubated with 400μ Ci of sodium ⁵¹ chromate for 30 min. at37° C. At the end of the incubation, the red blood cells are washedtwice with 20 mL HBSS to remove free sodium ⁵¹ chromate. The red bloodcells are finally reconstituted in 10 mL HBSS and 0.5 mL of the solution(about 20μ Ci) is injected per rat.

PROTEIN-LOSING GASTROPATHY IN SQUIRREL MONKEYS

Rationale

Protein-losing gastropathy (manifested as appearance of circulatingcells and plasma proteins in the GI tract) is a significant anddose-limiting adverse response to standard non-steroidalanti-inflammatory drugs (NSAIDs). This can be quantitatively assessed byintravenous administration of ⁵¹ CrCl₃ solution. This isotopic ion canavidly bind to cell and serum globins and cell endoplasmic reticulum.Measurement of radioactivity appearing in feces collected for 24 h afteradministration of the isotope thus provides a sensitive and quantitativeindex of protein-losing gastropathy.

Methods

Groups of male squirrel monkeys (0.8 to 1.4 kg) are treated by gavagewith either 1% methocell or 5% Tween 80 in H₂ O vehicles, (3 mL/kgb.i.d.) or test compounds at doses from 1-100 mg/kg b.i.d. for 5 days.Intravenous ⁵¹ Cr (5μ Ci/kg in 1 ml/kg phosphate buffer saline (PBS)) isadministered 1 h after the last drug/vehicle dose, and feces collectedfor 24 h in a metabolism cage and assessed for excreted ⁵¹ Cr bygamma-counting. Venous blood is sampled 1 h and 8 h after the last drugdose, and plasma concentrations of drug measured by RP-HPLC.

LPS-Induced Pyrexia in Conscious Rats

Male Sprague-Dawley rats (150-200 g) were fasted for 16-18 h before use.At approximately 9:30 a.m., the animals were placed temporarily inplexiglass restrainers and their baseline rectal temperature wasrecorded using a flexible temperature probe (YSI series 400) connectedto a digital thermometer (Model 08502, Cole Parmer). The same probe andthermometer were used for all animals to reduce experimental error. Theanimals were returned to their cages after the temperature measurements.At time zero, the rats were injected intraperitoneally with eithersaline or LPS (2 mg/kg, Sigma Chem) and the rectal temperature wasremeasured at 5, 6 and 7 h following LPS injection. After themeasurement at 5 h, when the increase in temperature had reached aplateau, the LPS-injected rats were given either the vehicle (1%methocel) or a test compound orally to determine whether the compoundcould reverse the pyrexia. Percent reversal of the pyrexia wascalculated using the rectal temperature obtained at 7 h in the control(vehicle-treated) group as the reference (zero reversal) point. Completereversal of pyrexia to the pre-LPS baseline value is taken as 100%.

LPS-Induced Pyrexia in Conscious Squirrel Monkeys

Temperature probes were surgically implanted under the abdominal skin ina group of squirrel monkeys (Saimiri sciureus) (1.0-1.7 kg). This allowsfor the monitoring of body temperature in conscious, unrestrainedmonkeys by a telemetric sensing system (Data Sciences International,Minnesota). The animals were fasted and were placed in individual cagesfor acclimatization 13-14 h before use. Electronic receivers wereinstalled on the side of the cages which pick up signals from theimplanted temperature probes. At approximately 9:00 a.m. on the day ofthe experiment, the monkeys were restrained temporarily in trainingchairs and were given a bolus I.V. injection of LPS, (6 mg/kg, dissolvedin sterile saline). The animals were returned to their cages and bodytemperature was recorded continuously every 5 min. Two h after injectionof LPS, when the body temperature had increased by 1.5-2 C, the monkeyswere dosed orally with either vehicle (1% methocel) or a test compound(3 mg/kg). One hundred minutes later, the difference between the bodytemperature and the baseline value was determined. Percent inhibitionwas calculated taking the value in the control group as 0% inhibition.

Acute Inflammatory Hyperalgesia Induced by Carrageenan in Rats

Experiments were performed using male Sprague Dawley rats (90-100 g).Hyperalgesia to mechanical compression of the hind paw was induced byintraplantar injection of carrageenan (4.5 mg into one hind paw) 3 hpreviously. Control animals received an equivalent volume of saline(0.15 ml intraplantar). A test compound (0.3-30 mg/kg, suspended in 0.5%methocel in distilled water) or vehicle (0.5% methocel) was administeredorally (2 ml/kg) 2 h after carrageenan. The vocalisation response tocompression of the hind paw was measured 1 h later using a Ugo Basilealgesiometer.

Statistical analysis for carrageenan-induced hyperalgesia was performedusing one-way ANOVA (BMDP Statistical Software Inc.). Hyperalgesia wasdetermined by subtracting the vocalisation threshold in saline injectedrats from that obtained in animals injected with carrageenan.Hyperalgesia scores for drug-treated rats were expressed as a percentageof this response. ID₅₀ values (the dose producing 50% of the maximumobserved response) were then calculated by nonlinear least squaresregression analysis of mean data using GraFit (Erithacus Software).

Adjuvant-Induced Arthritis in Rats

Seventy, 6.5-7.5 week old, female Lewis rats (body weight ˜146-170 g)were weighed, ear marked, and assigned to groups (a negative controlgroup in which arthritis was not induced, a vehicle control group, apositive control group administered indomethacin at a total daily doseof 1 mg/kg and four groups administered with a test compound at totaldaily doses of 0.10-3.0 mg/kg) such that the body weights wereequivalent within each group. Six groups of 10 rats each were injectedinto a hind paw with 0.5 mg of Mycobacterium butyricum in 0.1 ml oflight mineral oil (adjuvant), and a negative control group of 10 ratswas not injected with adjuvant. Body weights, contralateral paw volumes(determined by mercury displacement plethysmography) and lateralradiographs (obtained under Ketamine and Xylazine anesthesia) weredetermined before (day-1) and 21 days following adjuvant injection, andprimary paw volumes were determined before (day-1) and on days 4 and 21following adjuvant injection. The rats were anesthetized with anintramuscular injection of 0.03-0.1 ml of a combination of Ketamine (87mg/kg) and Xylazine (13 mg/kg) for radiographs and injection ofadjuvant. The radiographs were made of both hind paws on day 0 and day21 using the Faxitron (45 kVp, 30 seconds) and Kodak X-OMAT TL film, andwere developed in an automatic processor. Radiographs were evaluated forchanges in the soft and hard tissues by an investigator who was blindedto experimental treatment. The following radiographic changes weregraded numerically according to severity: increased soft issue volume(0-4), narrowing or widening of joint spaces (0-5) subchondral erosion(0-3), periosteal reaction (0-4), osteolysis (0-4) subluxation (0-3),and degenerative joint changes (0-3). Specific criteria were used toestablish the numerical grade of severity for each radiographic change.The maximum possible score per foot was 26. A test compound at totaldaily doses of 0.1, 0.3, 1, and 3 mg/kg/day, Indomethacin at a totaldaily dose of 1 mg/kg/day, or vehicle (0.5% methocel in sterile water)were administered per os b.i.d. beginning post injection of adjuvant andcontinuing for 21 days. The compounds were prepared weekly, refrigeratedin the dark until used, and vortex mixed immediately prior toadministration.

Two-factor (`treatment` and `time`) analysis of variance with repeatedmeasures on `time` were applied to the % changes for body weight andfoot volumes and to the rank-transformed radiographic total scores. Apost hoc Dunnett's test was conducted to compare the effect oftreatments to vehicle. A one-way analysis of variance was applied to thethymic and spleen weights followed by the Dunnett's test to compare theeffect of treatments to vehicle. Dose-response curves for % inhibitionin foot volumes on days 4, 14 and 21 were fitted by a 4-parameterlogistic function using a nonlinear least squares' regression. ID₅₀ wasdefined as the dose corresponding to a 50% reduction from the vehicleand was derived by interpolation from the fitted 4-parameter equation.

PHARMACOKINETICS IN RATS

Per Os Pharmacokinetics in Rats

The animals are housed, fed and cared for according to the Guidelines ofthe Canadian Council on Animal Care.

Male Sprague Dawley rats (325-375 g) are fasted overnight prior to eachPO blood level study.

The rats are placed in the restrainer one at a time and the box firmlysecured. The zero blood sample is obtained by nicking a small (1 mm orless) piece off the tip of the tail. The tail is then stroked with afirm but gentle motion from the top to the bottom to milk out the blood.Approximately 1 mL of blood is collected into a heparinized vacutainertube.

Compounds are prepared as required, in a standard dosing volume of 10mL/kg, and administered orally by passing a 16 gauge, 3" gavaging needleinto the stomach.

Subsequent bleeds are taken in the same manner as the zero bleed exceptthat there is no need to nick the tail again. The tail is cleaned with apiece of gauze and milked/stroked as described above into theappropriately labelled tubes.

Immediately after sampling, blood is centrifuged, separated, put intoclearly marked vials and stored in a freezer until analysed.

Typical time points for determination of rat blood levels after POdosing are:

0, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h

After the 4 hr time point bleed, food is provided to the rats adlibitum. Water is provided at all times during the study.

Vehicles

The following vehicles may be used in PO rat blood level determinations:

    ______________________________________                                        PEG 200/300/400    restricted to 2 mL/kg                                      Methocel 0.5%-1.0% 10 mL/kg                                                   Tween 80           10 mL/kg                                                   ______________________________________                                    

Compounds for PO blood levels can be in suspension form. For betterdissolution, the solution can be placed in a sonicator for approximately5 minutes.

For analysis, aliquots are diluted with an equal volume of acetonitrileand centrifuged to remove protein precipitate. The supernatant isinjected directly onto a C-18 HPLC column with UV detection.Quantitation is done relative to a clean blood sample spiked with aknown quantity of drug. Bioavailability (F) is assessed by comparingarea under the curve (AUC) i.v. versus p.o. ##EQU1## Clearance rates arecalculated from the following relation: ##EQU2## The units of CL aremL/h·kg (milliliters per hour kilogram)

Intravenous Pharmacokinetics in Rats

The animals are housed, fed and cared for according to the Guidelines ofthe Canadian Council on Animal Care.

Male Sprague Dawley (325-375 g) rats are placed in plastic shoe boxcages with a suspended floor, cage top, water bottle and food.

The compound is prepared as required, in a standard dosing volume of 1mL/kg.

Rats are bled for the zero blood sample and dosed under CO₂ sedation.The rats, one at a time, are placed in a primed CO₂ chamber and takenout as soon as they have lost their righting reflex. The rat is thenplaced on a restraining board, a nose cone with CO₂ delivery is placedover the muzzle and the rat restrained to the board with elastics. Withthe use of forceps and scissors, the jugular vein is exposed and thezero sample taken, followed by a measured dose of compound which isinjected into the jugular vein. Light digital pressure is applied to theinjection site, and the nose cone is removed. The time is noted. Thisconstitutes the zero time point.

The 5 min bleed is taken by nicking a piece (1-2 mm) off the tip of thetail. The tail is then stroked with a firm but gentle motion from thetop of the tail to the bottom to milk the blood out of the tail.Approximately 1 mL of blood is collected into a heparinized collectionvial. Subsequent bleeds are taken in the same fashion, except that thereis no need to nick the tail again. The tail is cleaned with a piece ofgauze and bled, as described above, into the appropriate labelled tubes.

Typical time points for determination of rat blood levels after I.V.dosing are either:

0, 5 min, 15 min, 30 min, 1 h, 2 h, 6 h

or

0, 5 min, 30 min, 1 h, 2 h, 4 h, 6 h.

Vehicles

The following vehicles may be used in IV rat blood level determinations:

    ______________________________________                                        Dextrose      1 mL/kg                                                         Moleculosol 25%                                                                             1 mL/kg                                                         DMSO (dimethylsulfoxide)                                                                    Restricted to a dose volume of 0.1 mL per                                     animal                                                          PEG 200       Not more than 60% mixed with 40%                                              sterile water-1 mL/kg                                           ______________________________________                                    

With Dextrose, either sodium bicarbonate or sodium carbonate can beadded if the solution is cloudy.

For analysis, aliquots are diluted with an equal volume of acetonitrileand centrifuged to remove protein precipitate. The supernatant isinjected directly onto a C-18 HPLC column with UV detection.Quantitation is done relative to a clean blood sample spiked with aknown quantity of drug. Bioavailability (F) is assessed by comparingarea under the curve (AUC) i.v. versus p.o. ##EQU3## Clearance rates arecalculated from the following relation: ##EQU4## The units of CL aremL/h·kg (milliliters per hour kilogram)

REPRESENTATIVE BIOLOGICAL DATA

Compounds of the present invention are inhibitors of COX-2 and arethereby useful in the treatment of COX-2 mediated diseases as enumeratedabove. The activities of the compounds against cyclooxygenase may beseen in the representative results shown below. In the assay, inhibitionis determined by measuring the amount of prostaglandin E₂ (PGE₂)synthesized in the presence of AA, COX-1 or COX-2 and a putativeinhibitor. The IC₅₀ values represent the concentration of putativeinhibitor required to lower PGE₂ synthesis to 50% of that obtained ascompared to the uninhibited control.

Data from three of these biological assays is given for representativecompounds along with comparative data for the following two compoundsfrom World Patent Application 96/10012:

                  TABLE 3                                                         ______________________________________                                         ##STR13##                                                                           Cox-2 Whole                                                                   Blood      Cox-1 U937                                                                              Selectivity                                                                          Rat Paw Edema                              Example                                                                              (IC.sub.50, μM)                                                                       (IC.sub.50, μM)                                                                      Ratio  (ED.sub.50, mg/kg)                         ______________________________________                                        Aa     7.9        >10       >1.3   >10                                        Ab     4.9        2.2       0.45   --                                         1      0.3        1.5       4.4    5.4                                        3      0.9        1.8       2      --                                         4      0.3        1         3.3    --                                         7      1.8        5         2.8    1.7                                        13     0.5        3         6      --                                         14     0.7        1.4       2      --                                         21     1.0        16        16     2.3                                        23     1.1        >10       >9.1   0.6                                        32     1.2        >10       >8.3   0.9                                        45     2.2        >10       >4.5   3.0                                        46                                 3.3                                        47                                 2.4                                        59                                 0.8                                        71     1.7        >10       >5.8   1.6                                        73     1.8        7         3.8    2.0                                        ______________________________________                                    

As can be seen from this data, the compounds of the present inventionshow greater COX-2 selectivity and potency than Aa and Ab. Moreover, thebasicity of the pyridine ring in these examples permits the formation ofacid salts, resulting in increased water solubility and gives thepotential for parenteral administration in aqueous vehicles.

The invention will now be illustrated by the following non-limitingexamples in which, unless stated otherwise:

(i) all operations were carried out at room or ambient temperature, thatis, at a temperature in the range 18-25° C. and drying of organics wasdone using MgSO₄,

(ii) evaporation of solvent was carried out using a rotary evaporatorunder reduced pressure (600-4000 pascals: 4.5-30 mm. Hg) with a bathtemperature of up to 60° C.,

(iii) the course of reactions was followed by thin layer chromatography(TLC) and reaction times are given for illustration only;

(iv) melting points are uncorrected and `d` indicates decomposition; themelting points given are those obtained for the materials prepared asdescribed; polymorphism may result in isolation of materials withdifferent melting points in some preparations;

(v) the structure and purity of all final products were assured by atleast one of the following techniques: TLC, mass spectrometry, nuclearmagnetic resonance (NMR) spectrometry or microanalytical data;

(vi) yields are given for illustration only;

(vii) when given, NMR data is in the form of delta (d) values for majordiagnostic protons, given in parts per million (ppm) relative totetramethylsilane (TMS) as internal standard, determined at 300 MHz or400 MHz using the indicated solvent; conventional abbreviations used forsignal shape are: s. singlet; d. doublet; t. triplet; m. multiplet; br.broad; etc.: in addition "Ar" signifies an aromatic signal;

(viii) chemical symbols have their usual meanings; the followingabbreviations have also been used v (volume), w (weight), b.p. (boilingpoint), m.p. (melting point), L (liter(s)), mL (milliliters), g(gram(s)), mg (milligrams(s)), mol (moles), mmol (millimoles), eq(equivalent(s)), r.t. (room temperature).

EXAMPLE 1

3-(4-Methylsulfonyl)phenyl-2-phenyl-5-trifluoromethylpyridine

Step 1: 2-Amino-3-bromo-5-trifluoromethylpyridine

To a solution of 2-amino-5-trifluoromethylpyridine (9 g) in acetic acid(75 mL) at r.t. was added bromine (5.8 mL) slowly. After 1 h, the acidwas neutralized by the careful addition of sodium hydroxide (10 N) at 0°C. The resulting orange precipitate was dissolved in ether and washedsuccessively with saturated potassium carbonate, saturated Na₂ SO₃ andbrine, dried and concentrated. The residual solid was stirred vigorouslyin hexane for 1 h to provide, after filtration, the title compound as awhite solid (10.2 g).

Step 2: 2-Amino-3-(4-methylthio)phenyl-5-trifluoromethylpyridine

A mixture of the bromide from Step 1, 4-methylthiobenzene boronic acid(Li, et. al. J. Med. Chem. 1995, 38, 4570) (8.5 g), 2 M aqueous sodiumcarbonate (60 mL) and palladium tetrakis(triphenylphosphine) (490 mg) inethanol/benzene (100 mL, 1:1) was heated at reflux for 15 h. The mixturewas cooled to r.t., diluted with water and extracted with ether. Theorganics were concentrated and the residue was subjected to stirredvigorously in ether/hexane for 1 h to provide, after filtration, thetitle compound (11.2 g) as a beige solid.

Step 3: 2-Amino-3-(4-methylsulfonyl)phenyl-5-trifluoromethylpyridine

A mixture of 2-amino-3-(4-methylthio)phenyl-5-trifluoromethylpyridine(9.7 g), OsO₄ (2 mL of a 4% solution in water) and NMO (13 g) inacetone/water (60 mL:5 mL) was stirred at r.t. overnight. Saturatedaqueous Na₂ SO₃ was then added and the resulting mixture was stirred for30 min. The acetone was evaporated and the resulting mixture wasextracted with ether and ethyl acetate. The combined organics werewashed with Na₂ SO₃, water, brine and then concentrated. The solidresidue was stirred vigorously in hexane and ether for 1 h and thenfiltered to provide the title compound as a pale yellow solid (9.9 g).

Step 4: 2-Chloro-3-(4-methylsulfonyl)phenyl-5-trifluoromethylpyridine

To a solution of2-amino-3-(4-methylsulfonyl)phenyl-5-trifluoromethylpyridine (1.2 g) inwater/concentrated HCl (9.5 mL:1 mL) at 0° C. was added a solution ofsodium nitrite (262 mg) in 5 mL water. The mixture was warmed to r.t.and stirred overnight. An additional 30 mg of sodium nitrite was addedand after 3 h the heterogeneous mixture was filtered. A portion of thesolid (250 mg) and POCl₃ (110 μL) in DMF (2 mL) was heated at 70° C. for60 h. The mixture was cooled to r.t., diluted with water and extractedwith ethyl acetate. The organics were washed with brine, dried andconcentrated to provide the title compound as a pale yellow solid (270mg) that was used as such in the subsequent reaction.

Step 5: 3-(4-Methylsulfonyl)phenyl-2-phenyl-5-trifluoromethylpyridine

A mixture of2-chloro-3-(4-methylsulfonyl)phenyl-5-trifluoromethylpyridine (260 mg),benzene boronic acid (113 mg), 2 M aqueous sodium carbonate (2.1 mL) andpalladium tetrakis(triphenylphosphine) (30 mg) in ethanol/benzene (8 mL,1:1) was heated at reflux for 24 h. The mixture was cooled to r.t.,diluted with water and extracted with ethyl acetate. The organics wereconcentrated and the solid residue was subjected to flash chromatography(eluting with hexane/ethyl acetate, 4:1 v/v) to provide the titlecompound as a white solid, m.p. 191-192° C. (215 mg).

EXAMPLE 2

2-(3-Chlorophenyl)-3-(4-methylsulfonyl)phenyl-5-trifluoromethylpyridine

Step 1: 2-Bromo-3-(4-methylsulfonyl)phenyl-5-trifluoromethylpyridine

To a solution of2-amino-3-(4-methylsulfonyl)phenyl-5-trifluoromethylpyridine fromExample 1, Step 3 (2 g) in 48% HBr (25 mL) at 0° C. was added bromine (3mL) and then sodium nitrite. (1.1 g) portionwise. After 2 h, thesolution was neutralized by the addition of sodium hydroxide (10 N) andthen extracted with ethyl acetate. The organics were washed withsaturated Na₂ SO₃ and brine, dried and concentrated. Flashchromatography of the residual material (eluting with hexane/ethylacetate, 7:3 to 3:7 v/v) provided the title compound as a white solid(435 mg).

Step 2:2-(3-Chlorophenyl)-3-(4-methylsulfonyl)phenyl-5-trifluoromethylpyridine

A mixture of2-bromo-3-(4-methylsulfonyl)phenyl-5-trifluoromethylpyridine (178 mg),3-chlorophenyl boronic acid (110 mg), potassium phosphate (225 mg) andpalladium tetrakis(triphenylphosphine) (20 mg) in dioxane (10 mL) washeated at reflux for 24 h. After cooling to room temperature, themixture was diluted with water and extracted with ether. The organicswere dried and concentrated and the residual material was subjected toflash chromatography (eluting with hexane/ethyl acetate, 7:3 v/v). Thesolid that was obtained was stirred vigorously in hexane/ether for 1 hto provide the title compound as a pale yellow solid, m.p. 136-237° C.(115 mg).

EXAMPLE 3

2-(4-Chlorophenyl)-3-(4-methylsulfonyl)phenyl-5-trifluoromethylpyridine

Following the procedures described in Example 1, Step 5, butsubstituting 4-chlorophenyl boronic acid for benzene boronic acid, thetitle compound was obtained as a white solid, m.p. 192-193° C. (155 mg).

EXAMPLE 4

2-(4-Fluorophenyl)-3-(4-methylsulfonyl)phenyl-5-trifluoromethylpyridine

Following the procedures described in Example 1, Step 5, butsubstituting 4-fluorophenyl boronic acid for benzene boronic acid, thetitle compound was obtained as a white solid, m.p. 163-164° C. (152 mg).

EXAMPLE 7

3-(4-Methylsulfonyl)phenyl-2-(3-pyridinyl)-5-trifluoromethylpyridine

A mixture of2-bromo-3-(4-methylsulfonyl)phenyl-5-trifluoromethylpyridine (600 mg)(Example 2, Step 2), diethyl-3-pyridinylborane (255 mg), sodiumcarbonate (2 M, 2.2 mL) and bis(triphenylphosphine)palladium dibromide(25 mg) in benzene/ethanol (1:1, 32 mL) was heated at reflux for 24 h.After cooling to r.t., the mixture was concentrated, diluted with waterand extracted with ethyl acetate. The organics were concentrated and theresidual material was dissolved in 10% HCl/ether. The organic phase wasremoved and the aqueous phase was adjusted to ˜pH 10 by the addition ofsaturated sodium bicarbonate. The mixture was extacted with ethylacetate and the combined organics were concentrated and subjected toflash chromatography (eluting with ethyl acetate) to provide the titlecompound as a white solid, m.p. 171-172° C. (180 mg).

EXAMPLE 13

5-Methyl-3-(4-methylsulfonyl)phenyl-2-phenylpyridine

Step 1: 2-Amino-3-bromo-5-methylpyridine

To a solution of 2-amino-5-picoline (5 g) in acetic acid (40 mL) at r.t.was added bromine (2.6 mL) slowly. After 1 h, the acid was neutralizedby the careful addition of sodium hydroxide (10 N) at 0° C. Theresulting orange precipitate was dissolved in ether and washedsuccessively with saturated potassium carbonate, saturated Na₂ S₂ O₃ andbrine, dried and concentrated. Flash chromatography (eluting withhexane/ethyl acetate, 3:2 v/v) of the residual solid provided the titlecompound as a pale yellow solid (7.1 g).

Step 2: 2-Amino-5-methyl-3-(4-methylsulfonyl)phenylpyridine

Following the procedures described in Example 1, Steps 2 and 3, butsubstituting 2-amino-3-bromo-5-methylpyridine (7.1 g) from Step 1 for2-amino-3-bromo-5-trifluoromethylpyridine, the title compound wasobtained as a pale yellow solid (3.7 g).

Step 3: 2-Bromo-5-methyl-3-(4-methylsulfonyl)phenylpyridine

Following the procedures described in Example 2, Step 1, butsubstituting 2-amino-5-methyl-3-(4-methylsulfonyl)phenyl-pyridine (3 g)from Step 2 for2-amino-3-(4-methylsulfonyl)phenyl-5-trifluoromethylpyridine, the titlecompound was obtained as a white solid (2.7 g).

Step 4: 5-Methyl-3-(4-methylsulfonyl)phenyl-2-phenylpyridine

Following the procedures described in Example 1, Step 5, butsubstituting 2-bromo-5-methyl-3-(4-methylsulfonyl)phenylpyridine (300mg) from Step 3 for2-chloro-3-(4-methylsulfonyl)phenyl-5-trifluoromethylpyridine, the titlecompound was obtained as a pale yellow solid (270 mg). ¹ H NMR (300 MHz,CDCl₃) δ 2.42 (s, 3H), 3.03 (s, 3H), 7.19-7.28 (m, 5H), 7.35 (d, 2H),7.51 (d, 1H), 7.81 (d, 2H), 8.56 (d, 1H).

EXAMPLE 14

2-(4-Chlorophenyl)-5-methyl-3-(4-methylsulfonyl)phenylpyridine

Following the procedures described in Example 3, but substituting2-bromo-5-methyl-3-(4-methylsulfonyl)phenylpyridine (300 mg) fromExample 13, Step 3 for2-chloro-3-(4-methylsulfonyl)phenyl-5-trifluoromethylpyridine, the titlecompound was obtained as a white solid, m.p. 155-156° C. (125 mg).

EXAMPLE 15

5-Methyl-3-(4-methylsulfonyl)phenyl-2-(3-pyridinyl)pyridine

Step 1: Lithium Tri-n-propoxy-3-pyridinylboronate

To a solution of 3-bromopyridine (39.5 g) in ether (800 mL) at -90° C.(internal temperature) was added n-BuLi (100 mL, 2.5 M) at a rate sothat the internal temperature did not exceed -78° C. The resultingmixture was stirred for 1 h at -78° C. and then triisopropoxyborate (59mL) was added and the resulting mixture was warmed to 0° C. Methanol wasadded and the mixture was evaporated three times from methanol and thentwo times from n-propanol. The residue was pumped under high vacuum for3 days and the resulting foam (76 g of a 1:1 mixture of the titlecompound:n-propanol) was used as such in the subsequent reaction.

Step 2: 5-Methyl-3-(4-methylsulfonyl)phenyl-2-(3-pyridinyl)pyridine

Following the procedures described in Example 14, but substitutinglithium tri-n-propoxy-3-pyridylboronate from Step 1 for 4-chlorophenylboronic acid, the title compound was obtained as a white solid, m.p.166-167° C. (2.1 g).

EXAMPLE 17

5-Chloro-2-(4-chlorophenyl)-3-(4-methylsulfonyl)phenylpyridine

Step 1: 2-Amino-3-bromo-5-chloropyridine

To a solution of 2-amino-5-chloropyridine (10 g) in acetic acid (75 mL)at r.t. was added bromine (2.6 mL) slowly. After 30 min, the acid wasneutralized by the careful addition of sodium hydroxide (10 N) at 0° C.The resulting orange precipitate was dissolved in ethyl acetate andwashed successively with saturated potassium carbonate, saturated Na₂ S₂O₃ and brine, dried and concentrated. Flash chromatography (eluting withhexane/ethyl acetate, 3:1 v/v) of the residual solid provided the titlecompound as a pale yellow solid (14.8 g).

Step 2: 2-Amino-5-chloro-3-(4-methylsulfonyl)phenylpyridine

Following the procedures described in Example 1, Steps 2 and 3, butsubstituting 2-amino-3-bromo-5-chloropyridine from Step 1 (5 g) for2-amino-3-bromo-5-trifluoromethylpyridine, the title compound wasobtained as a white solid (5 g).

Step 3: 2-Bromo-5-chloro-3-(4-methylsulfonyl)phenylpyridine

To a cold (ice bath) solution of2-amino-5-chloro-3-(4-methylsulfonyl)phenylpyridine (5 g) from Step 2 inwater/concentrated HCl (50 mL/10 mL) was added sodium nitrite (1.5 g) inwater (10 mL). The resulting mixture was stirred at r.t. for 15 h andthe resulting precipitate was removed by filtration and washedsuccessively with water and CCl₄. After air drying, the white solid (4.8g) and POBr₃ (10.5 g) in DMF (40 mL) were heated at 100° C. for 2 days.The resulting mixture was poured into ice/water and extracted with ethylacetate. The aqueous phase was made basic with 1 N NaOH and extractedwith ethyl acetate. The combined organics were dried and concentrated.Flash chromatography (eluting with hexane/ethyl acetate, 1:1 v/v) of theresidue provided the title compound as a white solid (2.7 g).

Step 4: 5-Chloro-2-(4-chloro)phenyl-3-(4-methylsulfonyl)phenylpyridine

Following the procedures described in Example 3 but substituting2-bromo-5-chloro-3-(4-methylsulfonyl)phenylpyridine (300 mg) from Step 3for 2-chloro-3-(4-methylsulfonyl)phenyl-5-trifluoromethylpyridine, thetitle compound was obtained as a white solid, m.p. 187-188° C. (200 mg).

EXAMPLE 20

5-Chloro-3-(4-methylsulfonyl)phenyl-2-(2-pyridinyl)pyridine

Step 1: 3-Bromo-5-chloro-2-hydroxypyridine

A mixture of 5-chloro-2-hydroxypyridine (100 g) and bromine (40.1 mL) inacetic acid (400 mL) was stirred at r.t. for 1 h. The mixture was pouredinto 3 L of water and stirred for 30 min then filtered. The residualsolid was washed with 2 L of cold water, air dried and then coevaporatedwith toluene three times and with benzene two times. The white solid (81g) so obtained was used in the subsequent reaction.

Step 2: 2-Benzyloxy-3-bromo-5-chloropyridine

A mixture of 3-bromo-5-chloro-2-hydroxypyridine (81 g), benzyl bromide(52 mL) and silver carbonate (97 g) in benzene (1 L) was heated at 70°C. for 1 h. The mixture was cooled to r.t. and then filtered through abed of celite. The filtrate was concentrated and the residual off-whitesolid was recrystallized from hexane to provide the title compound as awhite solid (102 g).

Step 3: 2-Benzyloxy-5-chloro-3-(4-methylsulfonyl)phenylpyridine

Following the procedures described in Example 1, Steps 2 and 3, butsubstituting 2-benzyloxy-3-bromo-5-chloropyridine (81 g) from Step 2 for2-amino-3-bromo-5-trifluoromethylpyridine, the title compound wasobtained as a white solid (72 g).

Step 4: 5-Chloro-2-hydroxy-3-(4-methylsulfonyl)phenylpyridine

A solution of 2-benzyloxy-5-chloro-3-(4-methylsulfonyl)phenylpyridine(72 g) in trifluoroacetic acid (250 mL) was stirred at 40° C. for 15 minand then poured into ice/water (˜1 L). After stirring for 10 min, thewhite solid was filtered, washed twice with a further 1 L of water andthen air dried to provide the title compound.

Step 5: 2,5-Dichloro-3-(4-methylsulfonyl)phenylpyridine

The crude 5-chloro-2-hydroxy-3-(4-methylsulfonyl)phenylpyridine fromStep 4 was heated in a sealed bomb at 150° C. with POCl₃ (400 mL) for 15h. After cooling to r.t. the excess POCl₃ was removed by distillationunder vacuum. The residue was diluted with ethyl acetate and water andthen neutralized with sodium hydroxide (10 N) to ˜pH 7. The organicswere removed, washed with brine and concentrated. The residual solid wasrecrystallized from ether to provide the title compound as white solid(61 g).

Step 6: Lithium Tri-n-propoxy-2-pyridylbornonate

Following the procedures described in Example 15, Step 1 butsubstituting 2-bromopyridine (1.9 mL) for 3-bromopyridine, the titlecompound was prepared as an off-white solid (4.1 g).

Step 7: 5-Chloro-3-(4-methylsulfonyl)phenyl-2-(2-pyridinyl)pyridine

A mixture of 2,5-dichloro-3-(4-methylsulfonyl)phenylpyridine (1 g),lithium tri-n-propoxy-2-pyridylboronate (1.22 g), sodium carbonate (5mL, 2 M) and bis(triphenylphosphine)palladium dibromide (520 mg) intoluene (100 mL), isopropanol (10 mL) and water (25 mL) was heated atreflux for 7 h. The mixture was cooled to r.t., diluted with ethylacetate and filtered through a bed of celite. The filtrated wasextracted with 6 N HCl and the aqueous was washed with ethyl acetate.The aqueous phase was basified to ˜pH 10 with 10 N sodium hydroxide andthen extracted with ethyl acetate. The organics were washed with brine,dried and concentrated. Flash chromatography (eluting with hexane/ethylacetate, 1:1 v/v) of the residue provided the title compound as a whitesolid, m.p. 134-135° C. (350 mg).

EXAMPLE 21

5-Chloro-3-(4-methylsulfonyl)phenyl-2-(3-pyridinyl)pyridine

Following the procedures described in Example 20, Step 7, butsubstituting lithium tri-n-propoxy-3-pyridinylboronate from Example 15,Step 1 for lithium tri-n-propoxy-2-pyridinylboronate, the title compoundwas obtained as a white solid, m.p. 168-169° C.

EXAMPLE 22

5-Chloro-3-(4-methylsulfonyl)phenyl-2-(4-pyridinyl)pyridine

Step 1: Lithium Trimethoxy-4-pyridinylbornonate

Following the procedures described in Example 15, Step 1 butsubstituting 4-bromopyridine for 3-bromopyridine. The crude material wasused prior to evaporating from n-propanol.

Step 2: 5-Chloro-3-(4-methylsulfonyl)phenyl-2-(4-pyridinyl)pyridine

Following the procedures described in Example 20, Step 7, butsubstituting lithium trimethoxy-4-pyridinylboronate from Step 1 forlithium tri-n-propoxy-2-pyridinylboronate, the title compound wasobtained as a white solid, m.p. 187-188° C.

EXAMPLE 23

5-Chloro-3-(4-methylsulfonyl)phenyl-2-(2-methyl-5-pyridinyl)pyridine

Step 1: Trifluoromethanesulfonic acid 2-methylpyridin-5-yl ester

To a mixture of 5-hydroxy-2-methylpyridine (2 g) and pyridine (1.9 mL)in dichloromethane (100 mL) at 0° C. was added trifluoromethanesulfonicacid anhydride (3.4 mL). The mixture was stirred at this temperature for15 min and then at r.t. for 45 min. Ammonium acetate (25%) was added andthe organics were removed and washed with 1 N HCl, dried andconcentrated. The title compound was obtained as a beige liquid (4 g)that was used as such.

Step 2: 2-Methyl-5-trimethylstannylpyridine

A mixture of trifluoromethanesulfonic acid 2-methylpyridin-5-yl ester(2.1 g), hexamethylditin (2.85 g), lithium chloride (1.1 g) andpalladium tetrakis(triphenylphosphine) (190 mg) was heated at reflux for180 min and then cooled to r.t. The mixture was filtered through a bedof celite, washing with ethyl acetate. The filtrate was washed twicewith 5% potassium fluoride, dried and concentrated. Flash chromatography(eluting with hexane/ethyl acetate, 6:1 v/v) of the residue provided thetitle compound as a pale yellow oil (1.3 g).

Step 3:5-Chloro-3-(4-methylsulfonyl)phenyl-2-(2-methyl-5-pyridyl)pyridine

A mixture of 2,5-dichloro-3-(4-methylsulfonyl)phenylpyridine fromExample 20, Step 5 (750 mg), 2-methyl-5-trimethylstannylpyridine (1.3 g)and palladium tetrakis(triphenylphosphine) (290 mg) in NMP (10 mL) washeated at 100° C. for 15 h. The mixture was cooled to r.t., diluted withethyl acetate and filtered through a bed of celite. The filtrate waswashed with water, twice with 5% potassium fluoride and then extractedwith 1 N HCl. The aqueous phase was neutralized with 10 N sodiumhydroxide and then extracted with ethyl acetate. The organics wereconcentrated and the residue subjected to flash chromatography (elutingwith ethyl acetate) to provide the title compound as a white solid, m.p.127-128° C.

EXAMPLE 43

2-(4-Chlorophenyl)-3-(4-methylsulfonyl)phenylpyridinyl-5-carboxylic acidmethyl ester

To a solution of2-(4-chlorophenyl)-5-methyl-3-(4-methylsulfonyl)phenylpyridine (Example14, 1.9 g) in t-butanol/water (1:2, 60 mL) at 90° C. was added solidpotassium permanganate (2.5 g) portionwise over 2 h. The resultingmixture was stirred at 90° C. for 15 h and then cooled to r.t. Themixture was filtered through a bed of celite and the filtrate wasacidified to ˜pH 2 with 6 N HCl. The white precipitate was filtered andthen a portion of this material was taken up in THF/dichloromethane.Diazomethane in ether was added to this solution until there was no morebubbling upon its addition. The resulting mixture was concentrated andsubjected to flash chromatography (eluting with hexane/ethyl acetate,1:1 v/v). The title compound was obtained as a white solid, m.p.216-218° C.

EXAMPLE 44

2-(4-Chlorophenyl)-3-(4-methylsulfonyl)phenylpyridinyl-5-carboxylic acid

To a solution of2-(4-chlorophenyl)-3-(4-methylsulfonyl)phenylpyridinyl-5-carboxylic acidmethyl ester (140 mg) in ethanol/water (1:1, 10 mL) was added lithiumhydroxide (0.35 mL, 3 N) and the resulting mixture was stirred at r.t.for 45 min. Saturated sodium bicarbonate was added and the aqueous wasextracted with ethyl acetate. The aqueous phase was treated with 3 N HCluntil ˜pH 2 and then was extracted with chloroform. The organics wereconcentrated to provide the title compound as a white solid, m.p. >300°C. (60 mg).

EXAMPLE 45

5-Cyano-2-(4-chlorophenyl)-3-(4-methylsulfonyl)phenylpyridine

To 2-(4-chlorophenyl)-3-(4-methylsulfonyl)phenylpyridinyl-5-carboxylicacid (300 mg) in dichloromethane (10 mL) at reflux was addedchlorosulfonylisocyanate (0.4 mL). After 90 min at reflux, the mixturewas cooled to r.t. and then DMF (1.5 mL) was added slowly. After 15 min,water was added and the mixture was extracted with ethyl acetate. Theorganics were washed with water, brine, dried and concentrated. Flashchromatography (eluting with ethyl acetate/hexane, 2:1 v/v) of theresidue provided the title compound as a white solid (100 mg). ¹ H NMR(300 MHz, CDCl₃) δ 3.06 (s, 3H), 7.21-7.28 (m, 4H), 7.37 (d, 2H), 7.90(d, 2H), 7.96 (d, 1H), 8.94 (d, 1H).

EXAMPLE 46

5-Chloro-3-(4-methylsulfonyl)phenyl-2-(3-pyridyl)pyridinehydromethanesulfonate

A solution of 5-chloro-3-(4-methylsulfonyl)phenyl-2-(3-pyridyl)pyridine(5.31 g, Example 21) in ethyl acetate (100 mL) was treated withmethanesulfonic acid (1 mL) in ethyl acetate (20 mL) dropwise. Theresulting precipitate was filtered and dried under vacuum to provide thetitle compound (6.4 g) as a white solid. ¹ H NMR (300 MHz, CD₃ OD) δ2.68 (s, 3H), 3.15 (s, 3H), 7.60 (d, 2H), 7.96-8.00 (m, 3H), 8.14 (d,1H), 8.47 (dt, 1H), 8.80 (d, 1H), 8.86 (m, 2H).

EXAMPLE 47

5-Chloro-3-(4-methylsulfonyl)phenyl-2-(3-pyridyl)pyridine hydrochloride

A solution of 5-chloro-3-(4-methylsulfonyl)phenyl-2-(3-pyridyl)pyridine(2.05 g, Example 21) in hot ethyl acetate (75 mL) was treated withhydrochloric acid (1.5 mL, 4 M in dioxane) dropwise. The resultingprecipitate was filtered and dried under vacuum to provide the titlecompound (2.2 g) as a white solid. ¹ H NMR (300 MHz, DMSO-d6) δ 3.24 (s,3H), 7.59 (d, 2H), 7.80 (dd, 1H), 7.91 (d, 2H), 8.15 (d, 1H), 8.22 (d,1H), 8.69 (d, 1H), 8.77 (d, 1H), 8.90 (d, 1H).

EXAMPLE 59

5-Chloro-3-(4-methylsulfonyl)phenyl-2-(2-methyl-5-pyridinyl)pyridineHydrochloride

Following the procedure described in Example 47, but substituting5-chloro-3-(4-methylsulfonyl)phenyl-2-(2-methyl-5-pyridinyl)pyridine(from Example 23) for5-chloro-3-(4-methylsulfonyl)phenyl-2-(3-pyridyl)pyridine, the titlecompound was obtained as a white solid. ¹ H NMR (300 MHz, DMSO-d₆): δ2.68 (s, 3H), 3.25 (s, 3H), 7.61 (d, 2H), 7.70 (d, 1H), 7.92 (d, 2H),8.07 (dd, 1H), 8.21 (d, 1H), 8.54 (d, 1H), 8.88 (d, 1H).

EXAMPLE 71

5-Chloro-3-(4-methylsulfonyl)phenyl-2-(2-ethyl-5-pyridinyl)pyridine##STR14##

Step 1: Lithium Tri-n-propoxy-2-ethyl-5-pyridylboronate

Following the procedures described in Example 15 Step 1 but substituting2-ethyl-5-bromopyridine (Tilley and Zawoiski, J. Org. Chem. 1988, 53,386) (4.6 g) for 3-bromopyridine, the title compound was prepared as ayellow solid.

Step 2:5-Chloro-3-(4-methylsulfonyl)phenyl-2-(2-ethyl-5-pyridinyl)pyridine

A mixture of 2,5-dichloro-3-(4-methylsulfonyl)phenylpyridine (Example 20Step 5) (5.6 g), lithium tri-n-propoxy-2-ethyl-5-pyridylboronate(Step 1) (4.0 g), sodium carbonate (17 mL, 2 M) andbis(triphenylphosphine)palladium dibromide (420 mg) in toluene (75 mL),ethanol (75 mL) and water (15 mL) was heated at reflux for 7 h. Themixture was cooled to r.t., diluted with ethyl acetate and filteredthrough a bed of celite. The filtrate was extracted with 6 N HCl and theaqueous was washed with ethyl acetate. The aqueous phase was basified to˜pH 10 with 10 N sodium hydroxide and then extracted with ethyl acetate.The organics were washed with brine, dried and concentrated. Flashchromatography (eluting with hexane/ethyl acetate, 1:1 v/v) of theresidue provided the title compound as a white solid (4.0 g). ¹ H NMR(500 MHz, acetone-d₆) δ 1.21 (t, 3H), 2.74 (q, 2H), 3.14 (s, 3H), 7.14,(d, 1H), 7.59 (m, 3H), 7.93 (d, 2H), 7.99 (d, 1H), 8.44 (d, 1H), 8.75(d, 1H).

EXAMPLE 71 Alternate Method

5-Chloro-3-(4-methylsulfonyl)phenyl-2-(2-ethyl-5-pyridinyl)pyridine##STR15##

Step 1: 5-Bromo-2-ethylpyridine

280 mL of 5 N sodium hydroxide (1.4 mol, 2.09 eq) was added to asolution of 158 g of 2,5-dibromopyridine (0.67 mol, 1 eq) in 1.4 L ofTHF. To the resulting solution, 700 mL of 1 N triethylboron in THF (0.70mol, 1.04 eq), 195 mg of bis(acetonitrile)palladium(II) chloride (0.75mmol, 0.0011 eq) and 414 mg 1,1'-bis(diphenylphosphino)ferrocene (0.75mmol, 0.0011 eq) were added. The reaction was slowly heated to a veryslight reflux for 3 hours. It was then cooled down to 0° and treatedsequentially with 140 mL of 5 N sodium hydroxide (0.70 mol, 1.04 eq) and53 mL of 30% hydrogen peroxide (0.70 mol, 1.05 eq) at such a rate thatthe temperature never exceeded 10° C. The mixture was extracted withether, and the ether extracts washed with sodium hydroxide, water, brineand dried over MgSO₄. The ether solution was then concentrated and thebrown residue was distilled under vacuum (40° C., 1 Torr) to give 112 gof the title compound as a clear oil slightly contaminated with thestarting material and 2,5-diethylpyridine. Yield ˜90%. The ¹ H NMR iscomparable to that reported by J. W. Tilley and S. Zawoiski; J. Org.Chem., 1988, 53, 386-390. The material is suitable for use in the nextstep without further purification.

Step 2: Lithium Tri-n-propoxy-2-ethyl-5-pyridylboronate

Following the procedures described in Example 15 Step 1 but substituting5-bromo-2-ethylpyridine from Step 1 for 3-bromopyridine, the titlecompound was prepared as a yellow solid.

Step 3:5-Chloro-3-(4-methylsulfonyl)phenyl-2-(2-ethyl-5-pyridinyl)pyridine

A mixture of 2,5-dichloro-3-(4-methylsulfonyl)phenylpyridine (Example 20Step 5) (5.6 g), lithium tri-n-propoxy-2-ethyl-5-pyridylboronate (Step2) (4.0 g), sodium carbonate (17 mL, 2 M) andbis(triphenylphosphine)palladium dibromide (420 mg) in toluene (75 mL),ethanol (75 mL) and water (15 mL) was heated at reflux for 7 h. Themixture was cooled to r.t., diluted with ethyl acetate and filteredthrough a bed of celite. The filtrate was extracted with 6 N HCl and theaqueous was washed with ethyl acetate. The aqueous phase was basified to˜pH 10 with 10 N sodium hydroxide and then extracted with ethyl acetate.The organics were washed with brine, dried and concentrated. Flashchromatography (eluting with hexane/ethyl acetate, 1:1 v/v) of theresidue provided the title compound as a white solid (4.0 g). ¹ H NMR(500 MHz, acetone-d₆) δ 1.21 (t, 3H), 2.74 (q, 2H), 3.14 (s, 3H), 7.14,(d, 1H), 7.59 (m, 3H), 7.93 (d, 2H), 7.99 (d, 1H), 8.44 (d, 1H), 8.75(d, 1H).

EXAMPLE 72

5-Chloro-3-(4-methylsulfonyl)phenyl-2-(2-ethyl-5-pyridinyl)pyridinehydromethanesulfonate ##STR16##

A solution of5-chloro-3-(4-methylsulfonyl)phenyl-2-(2-ethyl-5-pyridinyl)pyridine (3.4g, Example 71) in ethyl acetate (40 mL) and ether (100 mL) was treatedwith methanesulfonic acid (873 mg) in ether (20 mL) dropwise. Theresulting precipitate was cooled to -20° C., filtered and dried undervacuum to provide the title compound (4.3 g) as a white solid. ¹ H NMR(500 MHz, CD₃ OD) δ 1.40 (t, 3H), 2.68 (s, 3H), 3.07 (q, 2H), 3.15 (s,3H), 7.60 (d, 2H), 7.86 (d, 1H), 7.99 (d, 2H), 8.11 (d, 1H), 8.34 (m,1H), 8.69 (d, 1H), 8.83 (d, 1H).

EXAMPLE 73

5-Chloro-3-(4-methylsulfonyl)phenyl-2-(2-cyclopropyl-5-pyridinyl)pyridine

¹ H NMR (acetone-d₆) δ 0.9 (m, 4H), 2.0 (m, 1H), 3.14 (s, 3H), 7.15 (d,1H), 7.50 (dd, 1H), 7.59 (m, 2H), 7.95 (m, 3H), 8.36 (d, 1H), 8.72 (d,1H).

EXAMPLE 74

5-Chloro-3-(4-methylsulfonyl)phenyl-2-(2,6-dimethyl-3-pyridinyl)pyridine

    ______________________________________                                        Analysis for:                                                                            C             H      N                                             ______________________________________                                        Calculated 61.20         4.60   7.51                                          Found      61.52         4.52   7.87                                          ______________________________________                                    

ALTERNATIVE METHODS OF PREPARATION

Below are exemplified additional methods of preparation. The examplesare intended to be read independently. In particular, each example usesa compound numbering scheme that is internally consistant, but is notnecessarily consistant with the other examples or other portions of thespecification.

PREPARATION OF EXAMPLE 21

5-Chloro-3-(4-methylsulfonyl)phenyl-2-(3-pyridinyl)pyridine

Example 21

    ______________________________________                                         ##STR17##                                                                     ##STR18##                                                                    2-Chloromalondialdehyde                                                                            4.8 g (0.045 mol)                                        Ketone B             5.0 g (0.018 mol)                                        Propionic acid       30 mL                                                    Ammonium Acetate     8.4 g (0.11 mol)                                         ______________________________________                                    

A mixture of ketone B (5.0 g), 2-chloromalondialdehyde (4.8 g) andammonium acetate in propionic acid were heated to 130° C. The aceticacid produced was removed by distillation and heating continued at 136°C. for 15 hours. The reaction mixture was basified with sodiumcarbonate, water was added and the product was extracted intodichloromethane (2×150 mL). The organic layers were carbon treated(Dowex), dried (MgSO₄) and the solvent removed to afford 21 as an offwhite solid (3.4 g, 55% yield).

    ______________________________________                                         ##STR19##                                                                    2-Choromalondialdehyde                                                                            220 mg (2.1 mmol)                                         Oxalyl Chloride     180 mL (2.1 mmol)                                         Toluene             3 mL                                                      N,N-Dimethylformamide                                                                             20 mL                                                     ______________________________________                                    

N,N-dimethyl formamide was added to a slurry of 2-chloromalondialdehyde(220 mg) in toluene. Oxalyl chloride was added and the reaction mixturewas stirred until complete dissolution occurred.

    ______________________________________                                         ##STR20##                                                                     ##STR21##                                                                    Ketone B               500 mg (1.8 mmol)                                      Lithium bis(trimethylsilyl)amide (1 M in THF)                                                        1.8 mL (1.8 mmol)                                      Tetrahydrofuran        15 mL                                                  2,3-Dichloroacrolein in toluene                                                                      2.1 mmol in 3 mL                                       toluene                                                                       Ammonium acetate       1.0 g                                                  ______________________________________                                    

Lithium bis(trimethylsilyl)amide (1.8 mL; 1 M in THF) was added dropwiseto ketone B (500 mg) in THF (15 mL) at -78° C. The reaction mixture waswarmed to ambient temperature for 1 hour to form the lithium enolate ofB before recooling to -78° C. A solution of 2,3-dichloroacrolein wasadded and the temperature allowed to warm to room temperature. After 1hour ammonia gas was passed through the solution and after 30 minutesammonium acetate (1 g) was added. The reaction mixture was warmed to 60°C. for 1 hour and poured into aqueous sodium hydroxide (2 M; 100 mL).The product was extracted with dichloromethane (2×150 mL), dried (MgSO4)and the solvent removed to afford 21 (500 mg; 80%).

PREPARATION OF STARTING MATERIALS PREP 1

Synthesis of (4-methylsulfonyl)phenylacetic acid

    ______________________________________                                         ##STR22##                                                                     ##STR23##                                                                    Thioanisole 2 (FW = 124.2,                                                                        50.00 g (0.403 mol, 47.3 mL)                              d = 1.058)                                                                    Ethyloxalyl chloride (FW = 136.5,                                                                 82.43 g (0.604 mol, 67.5 mL)                              d = 1.222)                                                                    Aluminum chloride (FW = 133.3)                                                                    75.13 g (0.564 mol)                                       o-dichlorobenzene (ODCB)                                                                          112 mL                                                    ______________________________________                                    

The ethyloxalyl chloride and ODCB were charged to a flask equipped withan overhead mechanical stirrer and cooled to 0° C. The AlCl₃ was addedslowly. The addition of the AlCl₃ was exothermic. The thioanisole 2 wasadded dropwise via an addition funnel over 1.5 h. The reaction mixturerapidly turns a dark violet color. This addition was also exothermic.

After 1 h, the reaction was complete by HPLC. The reaction was quenchedby the slow addition of 300 mL of 1 N HCl at 0° C. After warming to roomtemperature, water and ODCB (50 mL each) were added. The layers weremixed and cut. The organic (bottom) phase was washed with 1×250 mL waterand then dried over MgSO₄.

This quench was also exothermic. The reaction mixture turned from darkviolet to pale green during the quench. The dried ODCB solution wascharged to a Morton flask equipped with mechanical stirring. A solutionof 1 N NaOH (800 mL) was added. The biphasic mixture was stirredvigorously and heated to 50° C. Hydrolysis to 3 was complete in 2-3 h byHPLC. The product-containing aqueous phase was taken directly into theWolf-Kishner reaction.

(4-Methylthio)phenylacetic acid

    ______________________________________                                         ##STR24##                                                                    3 (in 1N NaOH solution)                                                                           (0.402 mol)                                               Hydrazine (FW = 32.1, 35 wt % in water)                                                           206.14 g (2252 mol, 204 mL)                               NaOH (5N solution)  5 mL                                                      ______________________________________                                    

The hydrazine and NaOH were charged to a Morton flask equipped withmechanical stirring. After heating the hydrazine solution to 75° C., thesolution of 3 in NaOH was added over 35-40 min. At the end of theaddition the reaction mixture was brought to reflux for 5 days. HPLCshowed the reaction to be ca. 95% complete at this point. The startingmaterial was largely consumed in under 24 h, but a third peak appearedwhich took several days to convert to 4. The reaction was acidified withconcentrated HCl to pH=1.5 and extracted with EtOAc (1×750 mL and 1×250mL). The combined product-containing organic phases were washed 2×250 mL1 N HCl.

On acidification, the reaction mixture turned bright yellow.

(4-Methylsulfonyl)phenylacetic acid

    ______________________________________                                         ##STR25##                                                                     ##STR26##                                                                    (4-Methylthio)phenylacetic acid 4 (FW = 182.3)                                                        (0.402 mol)                                           Na.sub.2 WO.sub.4.H.sub.2 O (FW = 329.9)                                                              2.64 g (0.008 mol)                                    Aliquat 336 (FW = 404)  8.08 g (0.020 mol)                                    Hydrogen peroxide (FW = 34.0, 30 wt % in water)                                                       136 g (1.200 mol,                                     123 mL)                                                                       ______________________________________                                    

A flask equipped for mechanical stirring was charged with 4 (fromreaction above, in EtOAc), Aliquat 336, and Na₂ WO₄.2H₂ O (dissolved inca. 15 mL H₂ O). Hydrogen peroxide was added slowly via an additionfunnel over ca. 30 min. Completion of reaction was checked by HPLC. Thereaction was washed with 2×400 mL H₂ O and dried over MgSO₄.Quantification of product in the organic layer gave 61.29 g 5 (71% yieldfrom thioanisole). On concentration of the solution, a white solidprecipitated. The slurry was filtered, and washed with hexanes. Recoverywas 49.02 g 5 (57% from thioanisole).

Ivanov-Claisen Condensation for the Preparation of1-(3-pyridyl)-2-((4-methylsulfonyl)phenyl)ethane-1-one

    ______________________________________                                         ##STR27##                                                                     ##STR28##                                                                     ##STR29##                                                                    (4-Methylsulfonyl)benzyl-3-pyridylketone from ethyl nicotinate and (4-        methylsulfonyl)phenylacetic acid                                              ______________________________________                                        (4-Methylsulfonyl)phenyl Acetic Acid                                                               10 g (46.7 mmol)                                         (MW = 217)                                                                    t-Butyl magnesium chloride (1N/THF)                                                                128.11 ml (128.11 mmol)                                  Ethyl nicotinate (MW = 151.2; d = 1.107)                                                           5.54 ml (39.4 mmol)                                      THF                  400 ml                                                   ______________________________________                                    

(4-Methylsulfonyl)phenylacetic acid was dissolved in THF under nitrogenand 1.9 equivalents (88.73 ml) of t-butyl magnesium chloride were addedover 5 minutes to the solution. The reaction was exothermic and thetemperature rose from 20° C. to 50° C. After addition of the firstequivalent of t-butyl magnesium chloride, the solution turned red.

The reaction temperature was maintained at 50° C. After one hour, 0.5equivalents of ethyl nicotinate were added. The solution turned yellowand a white precipitate formed. After one hour, 0.5 equivalents oft-butyl magnesium chloride were added at 50° C. The solution turned red.This sequence of addition was repeated using 0.25 eq., 0.125 eq., 0.0625eq. of ethyl nicotinate and t-butyl magnesium chloride. The reactionmixture was aged for 1 hour between each addition.

After the last addition, the reaction was quenched by adding thereaction mixture into vigorously stirred 2 N hydrochloric acid (100 ml).The solids at the bottom of the reaction mixture dissolved witheffervescence when stirred in hydrochloric acid.

The pH of the aqueous phase of the reaction mixture was adjusted to 10with sodium carbonate. LC assay showed 91% yield of ketone

Preparation of (4-Methylsulfonyl)benzaldehyde

The preparation follows the procedure of Ulman JOC, pp4691 (1989).

(4-Methylsulfonyl)benzaldehyde from 4-fluorobenzaldehyde

    ______________________________________                                         ##STR30##                                                                

    ______________________________________                                        4-Fluorobenzaldehyde (MW = 124.11; d = 1.157)                                                            23.3 ml                                            (217 mmol)                                                                    Methanesulfinic acid, sodium salt (MW = 102.09) 24.23 g                                                  170 ml                                             (237 mmol) dimethyl sulfoxide                                                 ______________________________________                                    

Reagents were added to dimethyl sulfoxide and heated to 130° C. for 18hrs. The sodium methanesulfonate was partially insoluble at RT but wentinto solution at 130° C. Sodium fluoride precipitated out of solution.The reaction mixture was poured into 300 ml water. The productprecipitated out as a white solid. The reaction mixture was filtered.The product recovered was washed with 100 ml water and 2×50 ml methanolto remove dimethyl sulfoxide. The solvent was evaporated from theproduct under reduced pressure affording 39.9 g of the title compound asa white powder (86% isolated yield). C¹³ -NMR (CDCl₃): 44.33, 128.25,130.43, 139.70, 145.38, 190.72.

4-Methylsulfonylbenzaldehyde from 4-Chlorobenzaldehyde

    ______________________________________                                         ##STR31##                                                                

    ______________________________________                                        4-Chlorobenzaldehyde (MW = 140.57)                                                                    6.31 g (45 mmol)                                      Methanesulfinic acid, sodium salt (MW = 102.09)                                                       7.5 g (74 mmol)                                       dimethyl sulfoxide      50 ml                                                 ______________________________________                                    

Reagents were added to dimethyl sulfoxide and heated to 130° C. for 18hrs.

The sodium methanesulfinate was partially insoluble at RT but went intosolution at 130° C. Sodium chloride precipitated out of solution. Thereaction mixture was poured into 100 ml water. The product precipitatedout as a white solid. The reaction mixture was filtered. The productrecovered was washed with 50 ml water and 2×25 ml methanol to removedimethyl sulfoxide. The solvent was evaporated from the product underreduced pressure affording 5.1 g of (4-methylsulfonyl)benzaldehyde as awhite powder (62% isolated yield).

Homer/Wittig Route for the Preparation of1-(3-pyridyl)-2-((4-methylsulfonyl)phenyl)ethane-1-one

    ______________________________________                                         ##STR32##                                                                     ##STR33##                                                                    Ref: H. Zimmer, J. P. Bercz, Liebigs Ann. Chem. 1965, 686,                    ______________________________________                                        107-114.                                                                      Aniline              89.4 g (0.96 mol)                                        3-pyridinecarboxaldehyde                                                                           102.8 g (0.96 mol)                                       Ethanol              150 mL                                                   Diphenylphosphite    224.7 g (0.96 mol)                                       ______________________________________                                    

A solution of aniline in ethanol (50 mL) was added to a solution of3-pyridine carboxaldehyde in ethanol (100 mL) at 0° C. After 2 hoursdiphenylphosphite was added and stirring was continued at roomtemperature for 18 hours. Methyl tert-butylether (400 mL) was added tofurther precipitate the product which was filtered, washed (MTBE) anddried under vacuum to afford 320 g (80%) of the pyridylaminodiphenylphosphonate as a white solid. ¹³⁻ C NMR (CDCl3):

    ______________________________________                                         ##STR34##                                                                     ##STR35##                                                                    ______________________________________                                        Pyridyl-amino diphenylphosphonate                                                                     14.0 g (0.034 mol)                                    10% KOH in MeOH         23 mL (0.04 mol)                                      Tetrahydrofuran         150 mL                                                (4-methylsulfonyl)benzaldehyde                                                                        5.6 g (0.03 mol)                                      ______________________________________                                    

10% KOH/MeOH (23 mL) was added over 10 minutes to a solution of thephosphonate (14.0 g) in tetrahydrofuran at -45° C. After a further 10minutes the benzaldehyde was added in one portion and after 1 hour thereaction mixture was allowed to warm to ambient temperature. Aqueoushydrochloric acid (2 N, 100 mL) was added and the solution was leftstanding for 18 hours. EtOAc (200 mL) and water (200 mL) were added andthe organic layer discarded. The acid layer wash was basified (pH=9)with sodium carbonate and extracted with dichloromethane (2×150 mL). Theorganic layers were combined, dried (MgSO4) and concentrated.Trituration with hexanes afforded (4-methylsulfonyl)benzyl-3-pyridylketone as a pale yellow solid (6.3 g; 76%). ¹³⁻ C NMR (D-6 DMSO): 196.4,153.6, 149.4, 140.8, 139.1, 135.7, 131.5, 130.9, 126.8, 123.9, 44.6 and43.5 ppm.

    ______________________________________                                         ##STR36##                                                                     ##STR37##                                                                     ##STR38##                                                                    ______________________________________                                        Aniline               4.47 g (0.05 mol)                                       3-pyridinecarboxaldehyde                                                                            5.36 g (0.05 mol)                                       Methanol              10 mL                                                   Diphenylphosphite     11.2 g (0.05 mol)                                       10% KOH in MeOH       28 mL (0.05 mol)                                        (4-methylsulfonyl)benzaldehyde                                                                      8.3 g (0.45 mol)                                        ______________________________________                                    

A solution of aniline in methanol (5 mL) was added to a solution of3-pyridine carboxaldehyde in methanol (5 mL) at 0° C. After 2 hoursdiphenylphosphite was added and stirring was continued at roomtemperature for 18 hours. THF (100 mL) was added and the reaction wscooled to -40° C. 10% KOH/methanol (28 mL) was added and after 30minutes (4-methylsulfonyl)benzaldehyde (8.3 g) was added. The reactionwas allowed to warm to room temperature and stirred for 18 hours. EtOAc(200 mL) and water (200 mL) were added and the organic layer discarded.The acid layer was basified (pH=9) with sodium carbonate and extractedwith dichloromethane (2×150 mL). The organic layers were combined, dried(MgSO₄) and concentrated. Trituration with hexanes afforded(4-methylsulfonyl) benzyl-3-pyridyl ketone as a pale yellow solid (9.7g; 71%).

PREPARATION OF CHLOROMALONDIALDEHYDE

A number of routes are available for the preparation ofchloromalondialdehyde.

Preparation from 1,1,2,3,3-Pentachloropropane ##STR39##

A detailed experimental is published in Houben-Weyl-Muller: Methoden derOrganischen Chemie, 4th Edit., Vol 7/1, Thieme Verlag, Stuttgart, 1954,page 119. The starting material 1,1,2,3,3-pentachloropropane iscommercially available from Pfaltz and Bauer.

Preparation from Mucochloric Acid ##STR40## The following is a slightvariation of the original procedure of Dieckmann (Ber. Deut. Chem. Ges.1904, 37, 4638).

    ______________________________________                                        Mucochloric acid   50.0   g (0.30 mol)                                        Aniline            54     mL (0.60 mol)                                       Water              1000   mL                                                  ______________________________________                                    

To a solution of aniline in water at 85° C. in a vigorously stirred 2 Lflask was added mucochloric acid in small portions over 30 min. Onaddition of the mucochloric acid, a yellow color develops, which quicklydissipated. The reaction mixture stayed heterogeneous and filtration ofan aliquot after 30 min heating indicated completion of the reaction.

The reaction mixture was heated at 90° C. for 60 min., cooled to 50° C.and filtered. The filtercake was washed with 50 mL of 2 N HCl and 100 mLof H₂ O. The product was dried in a N₂ stream to give 57 g (100% yield)of 3-anilido-2-chloro-acrolein as a gray solid. ¹³ C NMR (D₆ -DMSO inppm): 108, 117, 124, 129, 140. 147, 182.

    ______________________________________                                        3-Anilido-2-chloro-acrolein                                                                         57 g (0.30 mol)                                         5N NaOH solution     120 mL (0.6 mol)                                         ______________________________________                                    

A solution of 3-anilido-2-chloro-acrolein in 120 mL of 5 N NaOH washeated to 100° C. for 90 min. The dark black solution was extractedtwice with 50 mL each of MTBE.

The first organic wash removed most of the dark color from the solution,and the second organic wash was only lightly colored.

On cooling the aqueous phase, a crystalline precipitate formed. Thisproduct was the 3-chloromalondialdehyde Na salt.

The aqueous phase was acidified by the addition of 60 mL of 37% HClsolution. The aqueous phase was extracted (MTBE/THF 50/50, 400 mL total)and the combined organic phases were dried over MgSO4. After treatmentwith Darco G60 and filtration through a plug of SiO2, the solution wasevaporated to give 19.6 g (62% overall yield) of chloromalondialdehydeas a dark solid. Recrystallization from ca. 10 mL of MTBE gave 11.13 gof pure chloromalondialdehyde as a tan solid. ¹³ C NMR (D₆ -DMSO inppm): 113, 175 (broad).

Preparation from Chloroacetylchloride ##STR41##

Arnold (Collect. Czech. Chem. Commun. 1961, 26, 3051) mentions theformation of 3-dimethylamino-2-chloro-acrolein by reaction ofchloroacetic acid with the Vilsmeyer reagent derived from POCl₃ and DMF.A variation and extension of his procedure prepareschloromalondialdehyde as its Na salt.

Oxalyl chloride (280 mL, 3.2 mol) was added at 10° C. to 1000 mL of DMF.The reaction was highly exothermic and a heavy precipitate formed. Aftera 2 h age, chloroacetylchloride (110 mL, 1.4 mol) was added and thereaction mixture was warmed to 75° C. for 3 hours. Analysis of analiquot by ¹ H NMR indicated complete consumption of thechloroacetylchloride and the reaction mixture was quenched by additioninto 1 L of H₂ O. To the cooled solution was added 500 mL of a 50% NaOHsolution. The reaction mixture was heated to reflux for 5 hours. Oncooling a precipitate formed, which was filtered and washed with water.The tan solid was dried in a N₂ stream to give 84 g of the sodium saltof chloromalondialdehyde as a tan solid (54% yield).

PREPARATION OF EXAMPLE 23

Step 1: Preparation of Weinreb amide

    ______________________________________                                         ##STR42##                                                                     ##STR43##                                                                    Materials         Amount    Mol     MW                                        ______________________________________                                        Methyl 6-methylnicotinate (1)                                                                   1.427 kg  9.439                                             151.2                                                                         N,O-Dimethylhydroxyainine.HCl                                                                   1.50 kg   15.38                                             97.6                                                                          Tetrahydrofuran   18 L                                                        Isopropylmagnesium chloride                                                                     12.8 L    25.60                                             (2.0 M in THF)                                                                Ammonium chloride 3.6 kg                                                      Water             18 L                                                        Hydrochloric acid 0.8 L                                                       Ethyl acetate     25 L                                                        Toluene           5 L                                                         ______________________________________                                    

Methyl 6-methylnicotinate (1) (1.42 kg, 9.44 mol),N,O-dimethylhydroxylamine.HCl (1.50 kg, 15.38 mol), and tetrahydrofuran(18 L) are added to a 100 L flask. The mixture is cooled to -20° C.

Isopropylmagnesium chloride (12.8 L, 25.6 mol) is added over 1 hmaintaining the temperature below -18° C.

The reaction is essentially complete within 25 min after the addition.The reaction mixture is added to aqueous ammonium chloride (20 wt %, 18L) at 5° C. in a 180 L Pflaubler.

The pH is adjusted to ˜7.0 (6.95) by the addition of concentratedhydrochloric acid (0.8 L) and the mixture is stirred for 20 min. Thelayers are separated.

The aqueous layer is extracted with ethyl acetate (2×10 L and 1×5 L).The organic layers (˜60 L) are concentrated under vacuum (23" Hg) at 50°C. Toluene (3 L) is added to the residue and the solution isconcentrated again. Solid impurities are removed by filtration through afrit and are washed with toluene (2 L). The filtrate is concentrated toafford the Weinreb amide 2 as a dark red oil (assay 1.78 kg, 92 wt%, >99% yield).

Step 2: DIBAL Reduction of Weinreb Amide 2 to 6-Methylnicotinaldehyde(3)

    ______________________________________                                         ##STR44##                                                                     ##STR45##                                                                    Materials:          Amount   Mol    MW                                        ______________________________________                                        Weinreb amide 2 (81 wt %)                                                                         5.09 kg  22.88  180.2                                     DIBAL (1.5 M in toluene)                                                                          17.0 L   25.50                                            Toluene             16 L                                                      Ethyl acetate       31 L                                                      L-Tartaric acid (20% w/v aqueous)                                                                 33 L                                                      50% Aqueous sodium hydroxide                                                                      2.3 L                                                     Silica Gel          866 g                                                     ______________________________________                                    

The Weinreb amide 2 (4.12 assay kg, 22.88 mol) is dissolved in toluene(16 L) in a 72 L glass, round-bottomed flask equipped with overheadstirring, a 5 L addition funnel, thermocouple probe, and a nitrogeninlet. The solution is cooled to -20° C. under nitrogen anddiisobutylaluminum hydride (17 L, 1.5 M in toluene, 25.5 mol) is addedover 2.5 h. The reaction temperature is maintained at <-5° C. during thecourse of the addition. When the addition is complete the reaction issampled.

Excess diisobutylaluminum hydride is quenched by the addition of ethylacetate (3.0 L). The reaction is aged for 30 min at -10° C. to 0° C. andthen added to a 20% tartaric acid solution (33 L) at 20° C.-25° C. over15 min using cooling to maintain the temperature <30° C.

The reaction vessel is rinsed with toluene (1.0 L) and the rinse isadded to the quench solution. The mixture is aged for 1 hour at 20-25°C. with efficient stirring. Two clear liquid phases are present. The pHis adjusted to 8.0 with 50% sodium hydroxide (2.3 L ) and the mixture isaged with stirring for another 30 min. The layers are separated and theaqueous layer is extracted with ethyl acetate (26 L).

The toluene layer (˜38 L) is concentrated under vacuum (29.5" Hg) at10-14° C. until the batch temperature reaches 30° C. The ethyl acetateextract is then added and the concentration is continued at 0-10° C.(29.5" Hg) to a final volume of ˜7 L. The remaining solution is filteredthrough silica gel (866 g, 230-400 mesh). The concentrating flask isrinsed with ethyl acetate (4 L) and the rinse is used to wash the cake.The cake is then washed with more ethyl acetate (6 L).

The combined filtrate and washes are transferred into a clean 22 L flaskthrough a 5 micron filter and concentrated under vacuum (29.5") at 0-5°C. The concentration is continued until the batch temperature reaches34° C. The liquid (4.08 kg, 60.8 wt %, 90% assay yield) is used as is inthe next step.

Step 3: Synthesis of the N,P-acetal 4

    ______________________________________                                         ##STR46##                                                                     ##STR47##                                                                    Materials           Amount   Mol     MW                                       ______________________________________                                        6-Methylnicotinaldehyde (3) (55 wt %)                                                             4.032 kg 18.02   124.1                                    Aniline (99%)       2.96 L   32.15   93.1                                     Diphenylphosphite (10-15% phenol)                                                                 7.6 L    ˜35.0                                                                           234.2                                    2-Propanol          15 L                                                      n-Heptane           60 L                                                      ______________________________________                                    

A 100 L flask is equipped with a mechanical stirrer, thermocouple, 5 Ldropping-funnel, and nitrogen inlet. 6-Methylnicotinaldehyde (2.238assay kg, 18.02 mol) is dissolved in 2-propanol (5 L). Aniline (2.96 L,32.15 mol, 1.8 equiv) is added in one portion (internal temperature: 16°C.) followed by the dropwise addition of diphenylphosphite (7.6 L, ˜35.0mol, ˜2 equiv) over 45 min. The diphenylphosphite contains 10-15%phenol.

Additional 2-propanol (5 L) is added followed by the slow addition ofn-heptane (40 L). The N,P-acetal 4 precipitates from solution

The mixture is cooled in an ice-bath at 5° C. The N,P-acetal is filteredand washed with n-heptane/2-propanol (4:1; 25 L). The resultingpale-white solid is dried for 24 h under low vacuum with a flow ofnitrogen affording 6.214 kg (90.7 wt %, 5.636 assay kg, 13.1 mol, 72.5%yield) of product.

Step 4: Synthesis of (4-Methylsulfonyl)benzaldehyde (6)

    ______________________________________                                         ##STR48##                                                                     ##STR49##                                                                    Materials         Amount    Mol    MW                                         ______________________________________                                        4-Fluorobenzaldehyde (5)                                                                        2500 g           20.1                                       124.1                                                                         Sodium methanesulfinate                                                                         2710 g           26.5                                       102.1                                                                         DMSO              5 L                                                         ______________________________________                                    

4-Fluorobenzaldehyde (2.5 kg, 20.1 mol) is dissolved in DMSO (5 L) andsodium methanesulfinate (2.71 kg, 26.5 mol) is added in one portion. Themixture is heated at 100° C.

The reaction is aged for 16 h or until complete. The reaction mixture ispoured into cold water (5° C.) (15 L) and aged for 1 h. The solid isfiltered and washed with water (10 L). The solid is dried under vacuumwith a nitrogen stream. The crude solid is mixed with isopropyl acetate(12 L) and the mixture is heated at 45° C. for 1 h. The slurry is cooledto 25° C. The solid is filtered, washed with isopropyl acetate andsuction dried under a nitrogen stream to afford 3.6 kg of the sulfone 6(97%).

Step 5: Synthesis of Ketosulfone 7

    ______________________________________                                         ##STR50##                                                                     ##STR51##                                                                     ##STR52##                                                                     ##STR53##                                                                    Materials          Amount    Mol    MW                                        ______________________________________                                        N,P-Acetal 4 (91 wt %)                                                                           6.21 kg   13.10  430.4                                     (4-Methylsulfonyl)benzaldehyde (6)                                                               2.68 kg   14.55  184.2                                     Cesium carbonate   5.72 kg   17.50  325.8                                     Tetrahydrofuran    20 L                                                       Absolute ethanol   5 L                                                        Hydrochloric acid  7.5 L                                                      Sodium Hydroxide (50%)                                                                           2.5 L                                                      Ethyl Acetate      45 L                                                       Hexane             34 L                                                       Water              56 L                                                       ______________________________________                                    

A 100 L flask is charged with tetrahydrofuran (20 L) and ethanol (5 L)followed by the N,P-acetal 4 (5.64 assay kg, 13.10 mol).(4-Methanesulfonyl)benzaldehyde (6) (2.68 kg, 14.55 mol) is added to thestirred solution.

Cesium carbonate (5.72 kg, 17.50 mol) is added in one portion.

The mixture is stirred until the reaction is complete.

Hydrochloric acid (14 L, 25%) is added over 1 h.

The reaction is stirred until the enamine hydrolysis is complete. Thereaction is complete in 2 h.

The solution is diluted with ethyl acetate (30 L) and extracted with 10%aqueous hydrochloric acid (2×12 L; 1×8 L; 1×6 L).

The aqueous extracts are combined and cooled in an ice/methanol bath.

Sodium hydroxide (50%, 2.5 L) is added over 30 min to adjust the pH to8, whereupon the ketosulfone precipitates.

The mixture is aged for 1 h and the temperature reaches 10° C. Theproduct is filtered, washed with water (10 L) and dried under a streamof nitrogen for 15 h. The solid (4.88 kg) is transferred to a 100 Lflask, and ethyl acetate (12 L) and hexanes (27 L) are added. Themixture is heated at 60° C. for 1 h, cooled, and aged at ambienttemperature for 3 h. The ketosulfone is filtered, washed withhexanes/ethyl acetate (7:3, 10 L) and dried under vacuum with a streamof nitrogen to afford 2.76 kg (98.3 A %, 97.9 wt %, 72% yield).

Step 6: Preparation of 3-Amino-2-Chloroacrolein (10)

    ______________________________________                                         ##STR54##                                                                     ##STR55##                                                                    Materials         Amount   Mol      MW                                        ______________________________________                                        Chloromalonaldehyde (8)                                                                         4.0 kg   37.6 mol 106.5                                     Aqueous ammonia (30%, 14.8 N)                                                                   3.70 L   54.8 mol 17.03                                     Isopropyl alcohol 64 L                                                        ______________________________________                                    

To a clean 22-L three-necked flask is charged chloromalonaldehyde (6)(4.0 kg, 37.6 mol) and isopropyl alcohol (4 L). The solution isconcentrated in a batch concentrator with a continuous, slow feed ofisopropyl alcohol (40 L total). The process requires 5 h to complete.The reaction is complete (>95% conversion) as judged by ¹ H NMR.

The resulting dark brown liquid is diluted with isopropyl alcohol (4 L).This mixture is then added over 2 min to a solution of 30% aqueousammonia (3.7 L) in isopropyl alcohol (20 L) cooled to 5° C. (ice bathcooling) in a 50 L round-bottomed flask.

The reaction solution is warmed to ambient and stirred for 18 h(overnight). The mixture is cooled down to 5° C. using ice bath (3 h).The product is filtered and dried under vacuum with a nitrogen stream toprovide 3.12 kg (78%) of the title compound. The filtrate isconcentrated to 3 L and the additional product is collected byfiltration and dried under vacuum with a nitrogen stream (0.61 kg, 15%).A total of 3.73 kg of product as a brown crystalline solid is obtained(93% overall yield).

Step 7: Preparation of Example 23

Step 7a: Cyclization of Ketosulfone and 3-Amino-2-chloroacrolein

    ______________________________________                                         ##STR56##                                                                     ##STR57##                                                                    Materials        Amount    Mol       MW                                       ______________________________________                                        3-Amino-2-chloroacrolein (10)                                                                  1.40 kg   13.1 mol  106.5                                    Ketosulfone 7 (98 wt %)                                                                        1.36 kg   4.69 mol  289.4                                    Methanesulfonic acid                                                                           0.992 kg  10.3 mol  96.10                                    n-Propionic acid 6.9 L     92.5 mol  74.08                                    Ammonium hydroxide (14.8 M)                                                                    6.0 L     88.8 mol  17.03                                    Toluene          13.5 L                                                       Isopropyl acetate                                                                              27 L                                                         ______________________________________                                    

To a solution of n-propionic acid (4 L) and toluene (8 L) at 75° C. in a50 L flask is charged ketosulfone 7 (1.385 kg, 4.69 mol) and3-amino-2-chloroacrolein (10) (1.40 kg, 13.1 mol). Propionic acid (2.9L, 92.5 mol total), methanesulfonic acid (0.67 L, 10.3 mol), and toluene(5.5 L) are then added. The resulting mixture is heated to reflux (114°C.). The reaction is completed in 13 h at 114° C. as judged by LC (97.5%conversion).

The reaction solution is cooled to ambient and diluted with isopropylacetate (10 L). Water (10 L) is added and the aqueous phase isneutralized with concentrated ammonium hydroxide solution (6.0 L) to pH7.2.

The resulting solution is transferred into a 100 L extraction flask, andadditional isopropyl acetate (8 L) and water (8 L) are added. Theorganic layer is washed with a 1:1 mixture of brine/water (2×10 L) andwater (10 L). The combined aqueous layers are extracted with isopropylacetate (9 L).

The combined organic layers are concentrated to a dark brown oil.

Step 7b: Purification of Example 23

Partial Purification of Example 23 Using Hot Extraction

    ______________________________________                                        Materials                                                                                         Amount                                                    ______________________________________                                               Ethyl acetate  24 L                                                           Hexanes        96 L                                                    ______________________________________                                    

The crude product from Step 7a is dissolved in ethyl acetate (12 L) in a72 L flask. The mixture is heated to 50° C., then hexane (48 L) is addedwhile the temperature is maintained at 50° C. The resulting solution isfiltered through a pad of Solka-floc (300 g) and the filtrate is setaside.

The Solk-floc cake is washed with ethyl acetate (6 L). This wash istransferred into the same 72 L flask and heated again to 50° C. Hexane(24 L) is added and this mixture is filtered at 50° C. The filtrate isset aside. Ethyl acetate (6 L) and hexane (24 L) are added to the 72 Lflask, The mixture is heated to 50° C. and filtered. All three filtratesare combined and concentrated to a brown oil in a batch concentrator.The residue is dissolved in methanol (4 L).

Example 23 HCl Salt Formation

    ______________________________________                                        Materials                                                                                   Amount   Mol     MW                                             ______________________________________                                        Hydrogen chloride in ether                                                                    3.2 L      3.2 mol 36.5                                       Ethyl acetate     5 L                                                         Methanol          5 L                                                         ______________________________________                                    

The methanol solution of Example 23 is further diluted with ethylacetate (5 L) and methanol (1 L). A solution of HCl in ether (1.0 M, 3.2L) is added over 5 min at 25-28° C.

The mixture is aged for 5 min at ambient temperature, cooled to 2° C.over 1 h, and aged for 2 h. The salt is filtered, washed with ethylacetate (2 L), and dried under vacuum with a nitrogen stream to provide1.17 kg of Example 23 as the hydrochloride salt (90 wt %, 95% recovery).

Salt Break and Final Crystallization

    ______________________________________                                        Materials                                                                                   Amount   Mol     MW                                             ______________________________________                                        30% Ammonium hydroxide                                                                        2.5 L      37 mol  17.03                                      Isopropyl acetate                                                                              62 L                                                         Hexanes          36 L                                                         ______________________________________                                    

Three batches of Example 23.HCl (3.37 kg, 90 wt %, 3.03 assayed kg, 7.67mol) are partitioned between isopropyl acetate (30 L) and water (30 L)in 100 L extraction flask. Concentrated ammonium hydroxide (2.5 L, 37mol) is added.

The separated organic phase is washed with water (2×15 L).

All three aqueous layers are back-extracted with isopropyl acetate (15L). The combined organic layers are treated with charcoal (600 g),stirred for 30 min, then filtered through a pad of Solka-Floc. Thefiltrate is concentrated, dissolved in isopropyl acetate (15 L) at 60°C. and passed through an in-line filter into a 72 L flask. Hexanes (15L) is added at 60° C., and after several minutes, the productcrystallizes. After 40 min additional hexanes (15 L) is added. Themixture is aged for 20 min, cooled from 43° C. to 15° C. over 2.5 h, to3° C. over 15 min, and aged at 3° C. for 30 min. The solid is filtered,washed with cold isopropyl acetate/hexanes (1:3; 8 L), and dried at roomtemperature under vacuum with a nitrogen stream to afford 1.92 kg ofExample 23.

What is claimed is:
 1. A compound of Formula I ##STR58## or apharmaceutically acceptable salt thereof wherein: R¹ is selected fromthe group consisting of:(a) CH₃, (b) NH₂, (c) NHC(O)CF₃ and (d) NHCH₃ ;Ar is a mono-, di-, or tri-substituted pyridinyl or pyridinyl-N-oxide,wherein the substituents are selected from the group consisting of:(a)hydrogen, (b) halo, (c) C₁₋₆ alkoxy, (d) C₁₋₆ alkylthio, (e) CN, (f)C₁₋₆ alkyl, (g) C₁₋₆ fluoroalkyl, (h) N₃, (i) --CO₂ R³, (j) hydroxy, (k)--C(R⁴)(R⁵)--OH, (l) --C₁₋₆ alkyl-CO₂ --R⁶ and (m) C₁₋₆ fluoroalkoxy; R²is selected from the group consisting of:(a) CN, (b) C₁₋₆ fluoroalkyl,(c) --CO₂ C₁₋₆ alkyl, (d) --C(R⁸)(R⁹)--OH and (e) --C₁₋₆ alkyl-CO₂ --R¹⁰; and R³, R⁴, R⁵, R⁶, R⁸, R⁹ and R¹⁰ are each independently selectedfrom the group consisting of:(a) hydrogen and (b) C₁₋₆ alkyl, or R⁴ andR⁵ or R⁸ and R⁹ together with the atom to which they are attached form asaturated monocyclic ring of 3, 4, 5, 6 or 7 atoms.
 2. A compound inaccordance with claim 1 wherein R² represents C1-6 fluoroalkyl.
 3. Acompound in accordance with claim 1 wherein R² represents CN.
 4. Acompound according to claim 2 of formula Ia ##STR59## or apharmaceutically acceptable salt thereof wherein R² and Ar are selectedin accordance with the following table:

    ______________________________________                                                   R.sup.2   Ar                                                       ______________________________________                                        (1)          CF.sub.3    3-pyr                                                (2)          CF.sub.3    5-(2-Me)pyr                                          (3)          CF.sub.3    5-(3-Br)pyr                                          (4)          CF.sub.3    5-(3-Cl)pyr                                          (5)          CF.sub.3    5-(2-OMe)pyr                                         (6)          CF.sub.3    2-(5-Br)pyr.                                         ______________________________________                                    


5. A compound according to claim 4 wherein the pharmaceuticallyacceptable salt is an acid salt of citric, hydrobromic, hydrochloric,maleic, methanesulfonic, phosphoric, sulfuric or tartaric acid.
 6. Acompound according to claim 1 of formula Ib ##STR60## or apharmaceutically acceptable salt thereof wherein: R² is CF₃ andAr is3-pyr.
 7. A compound according to claim 6 wherein the pharmaceuticallyacceptable salt is an acid salt of citric, hydrobromic, hydrochloric,maleic, methanesulfonic, phosphoric, sulfuric or tartaric acid.
 8. Acompound according to claim 2 whichis3-(4-Methylsulfonyl)phenyl-2-(3-pyridinyl)-5-trifluoromethylpyridine.9. A pharmaceutical composition for the treatment of cyclooxygenase-2mediated diseases, said composition comprising a compound according toclaim 1 and a pharmaceutically acceptable carrier.
 10. A compound offormula Ia ##STR61## or a pharmaceutically acceptable salt thereofwherein R² and Ar are selected in accordance with the following table:##STR62##
 11. A compound according to claim 10 wherein thepharmaceutically acceptable salt is an acid salt of citric, hydrobromic,hydrochloric, maleic, methanesulfonic, phosphoric, sulfuric or tartaricacid.
 12. A compound of formula Ib or a pharmaceutically acceptable saltthereof whereinR² and Ar are selected in accordance with the followingtable:

    ______________________________________                                                    R.sup.2 Ar                                                        ______________________________________                                        (1)           Cl        3-pyr                                                 (2)           Cl        5-(2-Me)pyr                                           (3)           Cl        5-(2-Et)pyr                                           (4)           Cl        5-(2-c-Pr)pyr                                         (5)           Cl        3-(2,6-Me.sub.2)pyr.                                  ______________________________________                                    


13. A compound according to claim 12 wherein the pharmaceuticallyacceptable salt is an acid salt of citric, hydrobromic, hydrochloric,maleic, methanesulfonic, phosphoric, sulfuric or tartaric acid.
 14. Acompound selectedfrom:5-Methyl-3-(4-methylsulfonyl)phenyl-2-(3-pyridinyl)pyridine;5-Chloro-3-(4-methylsulfonyl)phenyl-2-(2-pyridinyl)pyridine; and5-Chloro-3-(4-methylsulfonyl)phenyl-2-(4-pyridinyl)pyridine.
 15. Acompound according to claim 1 wherein Ar is a mono-, di-, ortrisubstituted 2-pyridinyl.
 16. A compound according to claim 1 whereinAr is a mono-, di-, or trisubstituted 3-pyridinyl.
 17. A compoundaccording to claim 1 wherein R¹ is CH₃ or NH₂.
 18. A compound accordingto claim 1 wherein Ar is a mono-, di-, or trisubstituted 2-pyridinyl or3-pyridinyl and the substituents are selected from the group consistingof:(a) hydrogen, (b) halo, (c) C₁₋₃ alkoxy, (d) C₁₋₃ alkylthio, (e) C₁₋₃alkyl, (f) CF₃ and (g) CN.
 19. A compound according to claim 1 whereinR¹ is CH₃ or NH₂ ; andAr is a mono-, di-, or trisubstituted 2-pyridinylor 3-pyridinyl and the substituents are selected from the groupconsisting of:(a) hydrogen, (b) halo, (c) C₁₋₃ alkoxy, (d) C₁₋₃alkylthio, (e) C₁₋₃ alkyl, (f) CF₃ and (g) CN.